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Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

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Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

What percentage of the patients were between 20 and 30 years old in this study?

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .

Why is EBC an attractive method for screening?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the leading cause of death among children after the age of 1 month?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How has the number of childhood pneumonia been reduced?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What percentage of childhood deaths are due to pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How has the childhood population grown in the last two decades?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the reduction in the number of childhood pneumonia cases?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How much is the reduction in the childhood pneumonia deaths?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Childhood pneumonia rate for high income countries vs low and middle income countries.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What percentage of childhood pneumonia deaths occur outside hospital in low and middle income countries?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Case Fatality Rates for Childhood Pneumonia in high income vs low and middle income countries.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How can childhood pneumonia affect the subsequent health of a person?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the increase in the risk of respiratory disease after having childhood pneumonia.

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Which is the best method to identify pneumonia in a person?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is responsible for the reduction of radiologic pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the percentage reduction in pneumonia cases due to vaccination?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the revised WHO definition of Bacterial Pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the reduction in bacterial pneumonia under the revised WHO definition of bacterial pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What caused the increase in the incidence of empyema in children in the recent past?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How have the incidence Empyema been reduced?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What pneumonia related or chest conditions indicate the need for child radiography?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What chest diseases and pneumonia were identified as leading causes prior to the availability of vaccines?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Why has pertussis immunity in infants has decreased in infants?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the effect of childhood tuberculosis in childhood pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What are the risk factors in childhood pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How does air pollution affect the incidence of childhood pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the strongest risk factor for childhood pneumonia?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is the global coverage of influenza and pneumonia vaccines?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

Is influenza vaccination during pregnancy safe? How long does it protect the child?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is emphyema?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

How is the term end point consolidation described with regard to pneumonia diagnosis?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

What factors make H5N1 a worldwide threat to public health?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

What are the symptoms of H5N1 infection in humans?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

Name some medications used to treat influenza.

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

Why have antiretrovirals medications had limited benefit in treating influenza?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

What is the focus of the current study?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

What is the result of the current study?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

How do the polysaccharides in plants effect the immune response?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

What does this study demonstrate?

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

In this study, how did treatment of EAP after infection affect survival?

A 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in Shenzhen, China https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181804/ SHA: ef5fe7296ec8baf90d974cf5737af0da3ed403ea Authors: He, Ying; Lin, Guang-Yu; Wang, Qiong; Cai, Xiao-Ying; Zhang, Yin-Hui; Lin, Chuang-Xing; Lu, Chang-Dong; Lu, Xue-Dong Date: 2014-05-14 DOI: 10.1111/irv.12257 License: cc-by Abstract: BACKGROUND: The epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. Limited data are available in China to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. OBJECTIVES: To determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in Shenzhen, China. METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality. RESULTS: Of the 2025 children enrolled in the study, 971 (48·0%) were positive for at least one viral pathogen, in which 890 (91·7%) were <4 years of age. The three most prevalent viruses were influenza A (IAV; 35·8%), respiratory syncytial virus (RSV; 30·5%) and human rhinovirus (HRV; 21·5%). Co-infections were found in 302 cases (31·1%), and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection. On the whole, the obvious seasonal peaks mainly from March to May were observed with peak strength varying from 1 year to another. CONCLUSIONS: This study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in Shenzhen. The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections (ARTIs) are a persistent and pervasive public health problem in both developed and developing countries. They cause a great burden of disease worldwide. Especially in developing countries including China, ARTIs, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus (RSV), influenza viruses A and B (IAV, IBV), parainfluenza viruses (PIVs), human rhinovirus (HRV) and adenovirus (ADV), which are responsible for most episodes of ARTIs in children. 1 In the past decade, several new viruses associated with ARTIs such as human metapneumovirus (HMPV), novel strains of coronaviruses (SARS-CoV, HCoV-NL63 and HKUI), human bocavirus (BOV), WU polyomavirus (WUPoyV) and KI polyomavirus (KIPoyV) have been discovered in human respiratory tract specimens. Among them, some have been identified to be causative pathogens of ARTIs. 1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs. Studies showed that many viral respiratory infections exhibited predictable seasonal variations. However, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. [6] [7] [8] [9] [10] [11] [12] China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. Although some studies on the epidemiology of ARTIs have recently been reported in big cities such as Beijing, Shanghai and Hong Kong, [13] [14] [15] [16] the epidemic characteristics of viruses in ARTIs are still not well established all around China, especially in other cities and rural areas. Shenzhen is the largest migratory city of China with high population density and population mobility. It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C (12-33°C) and 77%, respectively. The purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in Shenzhen and to provide insights into etiologies of ARTIs in local infants and children. A consecutive 3-year prospective study from July 2007 to June 2010 was conducted in Shenzhen, a coastal city neighboring Hong Kong. Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled. The inclusion criteria were as follows: less than 14 years old, acute fever (T ≥ 38°C), with any one of respiratory symptoms (such as sore throat, cough, wheezing and dyspnoea/ tachypnoea), normal or low leukocyte count, the onset of illness within 3 days before hospitalization. The diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia (CAP) issued by the Chinese Medical Association in 2006. 17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea (defined according to different age), difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary. The study protocol was approved by the medical ethical committees of the hospitals. Written informed consent was obtained from the parents or legal guardians of the children. Nasopharyngeal aspirates (NPA) were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing. Total viral nucleic acids (DNA and RNA) were extracted from 200 ll of NPA specimen using the AxyPrep Body Fluid DNA/RNA Miniprep Kit (Axygen, Union City, CA, USA), according to the manufacturer's instructions. Purified DNA and RNA were stored at À80°C in aliquots for further PCR analysis. For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously. 18, 19 Other viruses were tested using the Luminex platform and multiplex xTAG TM respiratory viral panel assay (RVP Assay) according to the manufacturer's instructions. 20 All multiple infection samples were retested. If there was discordance between two tests, the sample was confirmed by monoplex PCR. Statistical Package for the Social Sciences (SPSS) for Windows version 11.0 (SPSS Inc. Chicago, IL, USA) was used. For comparison of categorical data, chi-square or Fisher's exact test was used. All tests were two-tailed, and a P value below 0Á05 was considered statistically significant. A total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89Á6% of patients were < 4 years old. There were 964 (47Á6%) females and 1061 (52Á4%) males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection (Table 1) . Among 971 positive cases, 572 (58Á9%) were diagnosed as pneumonia. About 971 of the 2025 cases (48Á0%) were positive for at least one viral pathogen. Among them, IAV, RSV, HRV and PIVs were detected in 348 (35Á8%), 296 (30Á5%), 209 (21Á5%) and 169 (17Á4%) cases, respectively. Single infection was observed in 669 (68Á9%) cases, and multiple infection was found in 302 (31Á1%). Our results also showed that RSV, IAV and HRV were the main pathogens in single viral infection cases ( Table 1) . The monthly positive rates varied from 32Á5% to 75Á0% with a mean of 45Á1% ( Figure 1 ). In the year 2009, when influenza A (H1N1) was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected. The most frequently detected pathogen was IAV (26Á1%, 348/1335), followed by RSV (22Á2%, 296/1335), HRV (15Á7%, 209/1335), PIV1 and PIV3 (12Á7%, 169/1335) ( About 302 co-infection cases were identified, accounting for 14Á9% of all 2025 hospitalized children. During the H1N1 outbreak from March to August 2009, co-infection cases and co-infection rate increased significantly ( Figure 2 ). 143 of 302 (47Á4%) co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases. Of all co-infection cases, 247 (81Á8%), 49 (16Á2%) and 5 (1Á7%) were infected with two, three and four potential viral pathogens, respectively. One multiple infection with five viruses was detected in a RSV IAV HRV BOV PIV3 ADV HMPV WUPoyV PIV1 IBV Total A Total cases 163 172 85 55 51 49 37 26 24 7 669 Bronchitis 12 19 7 7 5 2 2 3 5 0 62 Bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 Pneumonia 93 97 55 28 24 28 25 18 14 5 387 URTI 13 37 3 13 9 10 2 3 0 2 *Case number and percentage in all enrolled children. **Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups. †No significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. IAV, RSV and HRV were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50Á6%, 44Á9% and 59Á3%, respectively ( Table 2) . Various multiple infection patterns were observed in the study. A total of 152 (50Á3%) co-infection cases involved at least two viruses of RSV, HRV and IAV. Co-infection rate of each individual virus detected varied significantly. The lowest and highest co-infection rates were observed in WUPoyV (33Á3%) and IBV (66Á7%), respectively. 91Á4% (276/302) of co-infection cases were tested in the age group of 4 years old or younger ( Table 2) , but among all age groups, no statistical difference in co-infection rate was found (v 2 = 1Á83, P = 0Á8721). Gender-specific difference in co-infection rate was not observed (v 2 = 2Á17, P = 0Á1404). There was no significant difference in co-infection rates between PICU and non-PICU cases. Similarly, no significant difference in clinical symptoms was observed between co-infection and single cases (data not shown). In general, respiratory viruses were detected more often in the period of March to May than in other months (55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000), and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years ( Figure 1) . The seasonality profile of each individual virus detected was diversified. A seasonal distribution of IAV can be observed from late spring to summer (mainly March to May) and sometimes in fall (October, November or December). A wide seasonal peak of IAV infection was detected from March to August 2009 ( Figure 3A ). Although RSV was tested almost a whole year, two yearly peaks were identified. One was found in November and/or December and the other stronger one was found in March to May of the year. The peak duration in 2009 was longer than those in other years. The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly ( Figure 3B ). Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. Peaks in spring and summer were also observed in some years ( Figure 3C ). Our investigation did not find regular seasonality in BOV infections. A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year. Of 39 patients with WUPoyV infection, 36 were detected after July 2008. Our data implied that peak months of WUPoyV infection were from March to May ( Figure 3D ). The positive rates of viral infections in male and female were 52Á5% and 47Á5%, respectively. No significant gender difference was revealed (v 2 = 0Á012, P = 0Á9118). The distribution of viral agents and infection patterns in different age groups are shown in Table 2 . Of all 971 positive children, 890 (91Á7%) were 4 years old or younger. The positive rate in this age group was significantly higher than that in children more than 4 years old (v 2 = 8Á26, P = 0Á0041). Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% (Table 2) . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children. In this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with ARTIs. Our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with ARTIs in the study areas, which was different from those in the big cities in northern China such as Beijing and Shanghai and also different from that in adjacent Hong Kong. Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies. [10] [11] [12] In China, the overall positive rate reported varied from 27Á3 to 74Á8% depending on different areas and detection methods. 15, 16, [25] [26] [27] [28] [29] [30] [31] The rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. Most studies showed that RSV or HRV was the most prevalent viruses in children with viral respiratory tract infection. 1 In this study, IAV was the most frequently detected respiratory virus, followed by RSV and HRV. IAV (H1N1) outbreak in 2009 could explain this shift. Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns. 24, 29, 33 Regardless of the IAV (H1N1) outbreak, RSV and HRV were the two most common viral pathogens in ARTIs, which was consistent with most previous studies. 1, 10, 15, 16, 22, [25] [26] [27] [28] [29] Our study further confirmed the importance of RSV and HRV in children with ARTIs, especially in children < 4 years of age. 10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome. 23 The prevalence of PIV3 was twofold higher than that of PIV1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with PIV3 than PIV1. HMPV has been proven to be one of the main viral pathogens responsible for ARTIs in children. 5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%. 22, 26, 28, 29, 31 The seasonality of HMPV in this study was mainly from March to May, similar to that in Hong Kong, 36 but different from other places. 5, 37 In our study, 4Á9% of cases were positive for BOV, which coincided with 5Á0% in Hong Kong 38 and higher than Guangzhou 39 and eastern Guangdong. 22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou. 39 The use of multiple PCR made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for ARTIs. 12,40 Among our positive cases, co-infection rate was 31Á1%, which was similar to 27Á9% reported by Do et al. 10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China. 22, [25] [26] [27] [28] [29] [30] [31] In most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. It was worth to note that in the study by Peng et al. 32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 Pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. It was notable that the correlation between co-infection rate and positive rate was not observed. Of multiple infections, dual infection was predominant in this study whether or not considering the IAV (H1N1) outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections. High co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. A variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 For example, it was shown in Martin et al.'s study 43 that ADV and coronaviruses were the most common co-infection pattern. Our study showed that RSV and HRV were the two most viruses involved in multiple infection, followed by IAV and PIVs, regardless of IAV infection in the H1N1 outbreak period. It was difficult to explain the variations of coinfection patterns based only on seasonal distribution. A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. Viral load detection could provide some clues for solving this issue. 43, 44 Although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of ARTIs were still not clear with inconsistent results in different studies. 42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al.'s study. 32 The relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. Several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded. Studies conducted in Hong Kong showed that a clear seasonal peak was from April to September, 36, 46 with a longer duration than our study. The overall seasonality in this study was also different from the studies conducted in northern or central cities of China, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, [25] [26] [27] 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in (sub-) tropical regions. In these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 Although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January (average 12°C). During the period from March to May, the weather featured warm ambient temperature (average 18-25°C), high relative humidity (average 85%-90%) and increasing rainfall. These meteorological conditions were perhaps conducive to viral survival. 9, 48 In addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children. This was particularly substantiated in RSV infection. Our presumption was supported by other studies. 14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study. Notably, our study occurred over a span of 3 years, which included the IAV (H1N1) outbreak in 2009. The impact of the outbreak on the results should be considered. Data showed that the detection rate of IAV increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV. Relatively higher single and multiple infections of RSV, HRV and PIVs were also observed during the outbreak of IAV. Increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. These factors could partly explain the relatively high proportion of pneumonia cases in the study. Furthermore, studies showed that the outbreak of IAV (H1N1) could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study. First, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. Second, the subtype identification of some common respiratory viruses such as IAV and HRV was not performed in our study, particularly during the IAV (H1N1) outbreak in 2009. In summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. It could help the prediction, prevention and control of ARTIs in children.

What viruses have been responsible for most common childhood acute respiratory track infections (ARTI)?

A 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in Shenzhen, China https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181804/ SHA: ef5fe7296ec8baf90d974cf5737af0da3ed403ea Authors: He, Ying; Lin, Guang-Yu; Wang, Qiong; Cai, Xiao-Ying; Zhang, Yin-Hui; Lin, Chuang-Xing; Lu, Chang-Dong; Lu, Xue-Dong Date: 2014-05-14 DOI: 10.1111/irv.12257 License: cc-by Abstract: BACKGROUND: The epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. Limited data are available in China to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. OBJECTIVES: To determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in Shenzhen, China. METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality. RESULTS: Of the 2025 children enrolled in the study, 971 (48·0%) were positive for at least one viral pathogen, in which 890 (91·7%) were <4 years of age. The three most prevalent viruses were influenza A (IAV; 35·8%), respiratory syncytial virus (RSV; 30·5%) and human rhinovirus (HRV; 21·5%). Co-infections were found in 302 cases (31·1%), and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection. On the whole, the obvious seasonal peaks mainly from March to May were observed with peak strength varying from 1 year to another. CONCLUSIONS: This study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in Shenzhen. The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections (ARTIs) are a persistent and pervasive public health problem in both developed and developing countries. They cause a great burden of disease worldwide. Especially in developing countries including China, ARTIs, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus (RSV), influenza viruses A and B (IAV, IBV), parainfluenza viruses (PIVs), human rhinovirus (HRV) and adenovirus (ADV), which are responsible for most episodes of ARTIs in children. 1 In the past decade, several new viruses associated with ARTIs such as human metapneumovirus (HMPV), novel strains of coronaviruses (SARS-CoV, HCoV-NL63 and HKUI), human bocavirus (BOV), WU polyomavirus (WUPoyV) and KI polyomavirus (KIPoyV) have been discovered in human respiratory tract specimens. Among them, some have been identified to be causative pathogens of ARTIs. 1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs. Studies showed that many viral respiratory infections exhibited predictable seasonal variations. However, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. [6] [7] [8] [9] [10] [11] [12] China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. Although some studies on the epidemiology of ARTIs have recently been reported in big cities such as Beijing, Shanghai and Hong Kong, [13] [14] [15] [16] the epidemic characteristics of viruses in ARTIs are still not well established all around China, especially in other cities and rural areas. Shenzhen is the largest migratory city of China with high population density and population mobility. It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C (12-33°C) and 77%, respectively. The purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in Shenzhen and to provide insights into etiologies of ARTIs in local infants and children. A consecutive 3-year prospective study from July 2007 to June 2010 was conducted in Shenzhen, a coastal city neighboring Hong Kong. Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled. The inclusion criteria were as follows: less than 14 years old, acute fever (T ≥ 38°C), with any one of respiratory symptoms (such as sore throat, cough, wheezing and dyspnoea/ tachypnoea), normal or low leukocyte count, the onset of illness within 3 days before hospitalization. The diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia (CAP) issued by the Chinese Medical Association in 2006. 17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea (defined according to different age), difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary. The study protocol was approved by the medical ethical committees of the hospitals. Written informed consent was obtained from the parents or legal guardians of the children. Nasopharyngeal aspirates (NPA) were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing. Total viral nucleic acids (DNA and RNA) were extracted from 200 ll of NPA specimen using the AxyPrep Body Fluid DNA/RNA Miniprep Kit (Axygen, Union City, CA, USA), according to the manufacturer's instructions. Purified DNA and RNA were stored at À80°C in aliquots for further PCR analysis. For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously. 18, 19 Other viruses were tested using the Luminex platform and multiplex xTAG TM respiratory viral panel assay (RVP Assay) according to the manufacturer's instructions. 20 All multiple infection samples were retested. If there was discordance between two tests, the sample was confirmed by monoplex PCR. Statistical Package for the Social Sciences (SPSS) for Windows version 11.0 (SPSS Inc. Chicago, IL, USA) was used. For comparison of categorical data, chi-square or Fisher's exact test was used. All tests were two-tailed, and a P value below 0Á05 was considered statistically significant. A total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89Á6% of patients were < 4 years old. There were 964 (47Á6%) females and 1061 (52Á4%) males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection (Table 1) . Among 971 positive cases, 572 (58Á9%) were diagnosed as pneumonia. About 971 of the 2025 cases (48Á0%) were positive for at least one viral pathogen. Among them, IAV, RSV, HRV and PIVs were detected in 348 (35Á8%), 296 (30Á5%), 209 (21Á5%) and 169 (17Á4%) cases, respectively. Single infection was observed in 669 (68Á9%) cases, and multiple infection was found in 302 (31Á1%). Our results also showed that RSV, IAV and HRV were the main pathogens in single viral infection cases ( Table 1) . The monthly positive rates varied from 32Á5% to 75Á0% with a mean of 45Á1% ( Figure 1 ). In the year 2009, when influenza A (H1N1) was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected. The most frequently detected pathogen was IAV (26Á1%, 348/1335), followed by RSV (22Á2%, 296/1335), HRV (15Á7%, 209/1335), PIV1 and PIV3 (12Á7%, 169/1335) ( About 302 co-infection cases were identified, accounting for 14Á9% of all 2025 hospitalized children. During the H1N1 outbreak from March to August 2009, co-infection cases and co-infection rate increased significantly ( Figure 2 ). 143 of 302 (47Á4%) co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases. Of all co-infection cases, 247 (81Á8%), 49 (16Á2%) and 5 (1Á7%) were infected with two, three and four potential viral pathogens, respectively. One multiple infection with five viruses was detected in a RSV IAV HRV BOV PIV3 ADV HMPV WUPoyV PIV1 IBV Total A Total cases 163 172 85 55 51 49 37 26 24 7 669 Bronchitis 12 19 7 7 5 2 2 3 5 0 62 Bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 Pneumonia 93 97 55 28 24 28 25 18 14 5 387 URTI 13 37 3 13 9 10 2 3 0 2 *Case number and percentage in all enrolled children. **Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups. †No significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. IAV, RSV and HRV were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50Á6%, 44Á9% and 59Á3%, respectively ( Table 2) . Various multiple infection patterns were observed in the study. A total of 152 (50Á3%) co-infection cases involved at least two viruses of RSV, HRV and IAV. Co-infection rate of each individual virus detected varied significantly. The lowest and highest co-infection rates were observed in WUPoyV (33Á3%) and IBV (66Á7%), respectively. 91Á4% (276/302) of co-infection cases were tested in the age group of 4 years old or younger ( Table 2) , but among all age groups, no statistical difference in co-infection rate was found (v 2 = 1Á83, P = 0Á8721). Gender-specific difference in co-infection rate was not observed (v 2 = 2Á17, P = 0Á1404). There was no significant difference in co-infection rates between PICU and non-PICU cases. Similarly, no significant difference in clinical symptoms was observed between co-infection and single cases (data not shown). In general, respiratory viruses were detected more often in the period of March to May than in other months (55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000), and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years ( Figure 1) . The seasonality profile of each individual virus detected was diversified. A seasonal distribution of IAV can be observed from late spring to summer (mainly March to May) and sometimes in fall (October, November or December). A wide seasonal peak of IAV infection was detected from March to August 2009 ( Figure 3A ). Although RSV was tested almost a whole year, two yearly peaks were identified. One was found in November and/or December and the other stronger one was found in March to May of the year. The peak duration in 2009 was longer than those in other years. The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly ( Figure 3B ). Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. Peaks in spring and summer were also observed in some years ( Figure 3C ). Our investigation did not find regular seasonality in BOV infections. A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year. Of 39 patients with WUPoyV infection, 36 were detected after July 2008. Our data implied that peak months of WUPoyV infection were from March to May ( Figure 3D ). The positive rates of viral infections in male and female were 52Á5% and 47Á5%, respectively. No significant gender difference was revealed (v 2 = 0Á012, P = 0Á9118). The distribution of viral agents and infection patterns in different age groups are shown in Table 2 . Of all 971 positive children, 890 (91Á7%) were 4 years old or younger. The positive rate in this age group was significantly higher than that in children more than 4 years old (v 2 = 8Á26, P = 0Á0041). Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% (Table 2) . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children. In this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with ARTIs. Our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with ARTIs in the study areas, which was different from those in the big cities in northern China such as Beijing and Shanghai and also different from that in adjacent Hong Kong. Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies. [10] [11] [12] In China, the overall positive rate reported varied from 27Á3 to 74Á8% depending on different areas and detection methods. 15, 16, [25] [26] [27] [28] [29] [30] [31] The rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. Most studies showed that RSV or HRV was the most prevalent viruses in children with viral respiratory tract infection. 1 In this study, IAV was the most frequently detected respiratory virus, followed by RSV and HRV. IAV (H1N1) outbreak in 2009 could explain this shift. Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns. 24, 29, 33 Regardless of the IAV (H1N1) outbreak, RSV and HRV were the two most common viral pathogens in ARTIs, which was consistent with most previous studies. 1, 10, 15, 16, 22, [25] [26] [27] [28] [29] Our study further confirmed the importance of RSV and HRV in children with ARTIs, especially in children < 4 years of age. 10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome. 23 The prevalence of PIV3 was twofold higher than that of PIV1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with PIV3 than PIV1. HMPV has been proven to be one of the main viral pathogens responsible for ARTIs in children. 5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%. 22, 26, 28, 29, 31 The seasonality of HMPV in this study was mainly from March to May, similar to that in Hong Kong, 36 but different from other places. 5, 37 In our study, 4Á9% of cases were positive for BOV, which coincided with 5Á0% in Hong Kong 38 and higher than Guangzhou 39 and eastern Guangdong. 22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou. 39 The use of multiple PCR made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for ARTIs. 12,40 Among our positive cases, co-infection rate was 31Á1%, which was similar to 27Á9% reported by Do et al. 10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China. 22, [25] [26] [27] [28] [29] [30] [31] In most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. It was worth to note that in the study by Peng et al. 32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 Pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. It was notable that the correlation between co-infection rate and positive rate was not observed. Of multiple infections, dual infection was predominant in this study whether or not considering the IAV (H1N1) outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections. High co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. A variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 For example, it was shown in Martin et al.'s study 43 that ADV and coronaviruses were the most common co-infection pattern. Our study showed that RSV and HRV were the two most viruses involved in multiple infection, followed by IAV and PIVs, regardless of IAV infection in the H1N1 outbreak period. It was difficult to explain the variations of coinfection patterns based only on seasonal distribution. A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. Viral load detection could provide some clues for solving this issue. 43, 44 Although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of ARTIs were still not clear with inconsistent results in different studies. 42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al.'s study. 32 The relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. Several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded. Studies conducted in Hong Kong showed that a clear seasonal peak was from April to September, 36, 46 with a longer duration than our study. The overall seasonality in this study was also different from the studies conducted in northern or central cities of China, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, [25] [26] [27] 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in (sub-) tropical regions. In these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 Although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January (average 12°C). During the period from March to May, the weather featured warm ambient temperature (average 18-25°C), high relative humidity (average 85%-90%) and increasing rainfall. These meteorological conditions were perhaps conducive to viral survival. 9, 48 In addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children. This was particularly substantiated in RSV infection. Our presumption was supported by other studies. 14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study. Notably, our study occurred over a span of 3 years, which included the IAV (H1N1) outbreak in 2009. The impact of the outbreak on the results should be considered. Data showed that the detection rate of IAV increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV. Relatively higher single and multiple infections of RSV, HRV and PIVs were also observed during the outbreak of IAV. Increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. These factors could partly explain the relatively high proportion of pneumonia cases in the study. Furthermore, studies showed that the outbreak of IAV (H1N1) could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study. First, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. Second, the subtype identification of some common respiratory viruses such as IAV and HRV was not performed in our study, particularly during the IAV (H1N1) outbreak in 2009. In summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. It could help the prediction, prevention and control of ARTIs in children.

Are there any vaccines against to protect against respiratory viral infections?

Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377511/ SHA: 615071c8c959f24857b1bad521cc432b59719bfb Authors: Kotay, Shireen; Chai, Weidong; Guilford, William; Barry, Katie; Mathers, Amy J. Date: 2017-03-31 DOI: 10.1128/aem.03327-16 License: cc-by Abstract: There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports (1) (2) (3) (4) (5) , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link (6) (7) (8) (9) (10) (11) (12) (13) . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents (14, 15) . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap (6, (16) (17) (18) (19) (20) . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities (16, 21, 22) . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed (23) (24) (25) (26) (27) (28) . There are many reports of a genetic association between pathogens found in sink traps and those found in patients (29, 30) . However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles (Glo Germ; Glo Germ Co., Moab, UT) that material injected into the P-trap gets dispersed around a hand-washing sink (6) . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: (i) can organisms grow retrograde from the P-trap water to the sink strainer, (ii) can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and (iii) which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing (30) . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein (GFP)-expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth (TSB) promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap (Table 1) . On day 7, the strainer (ϳ8 in. above the liquid in the P-trap) was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink (sink 5) was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration (10 3 CFU/ml) in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps (Fig. 1a) . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days (Fig. 1b and c) . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen (described previously) was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space (Fig. 2) . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl (zones 2, 5, 6, 9, 11, and 12) . Anterior corners of the sink counter space (zones 4 and 7), which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres (Fig. 2) . Dispersion was significantly higher along the axes (zones 6, 9, 11, and 12) and lower at the corners of the sink counter space (zones 4, 7, and 10). In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl (Fig. 3b) . Further, dispersion was greater along the Ϫ" and "ϩ" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl (Fig. 3b , zones B3, B4, and B10) than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets (Fig. 3a , zones 12 and 11). A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space (Fig. 3a , zones 4, 7, and 10) was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface (Fig. 3c ). There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done (6, 28) . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission (29, 31, 32) . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms (3, 33) . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles (6) . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported (6) . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks (intravenous fluids, feeding supplements, and leftover beverages) (5, 32) . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing (23) . Sink 3 was lowest on the slope in the drain line (see Fig. 4 ) with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source (sink 5), and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome (SARS) and Ebola viruses through premise plumbing systems (34) (35) (36) . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies (7, 10, 13, 24, 37) was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water (17, 20) . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal (6) . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users (38) . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other (Fig. 4) . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet (Elkay, Oak Brook, IL). The drain line (Dearborn Brass-Oatey, Cleveland, OH). All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 (Fig. 4) . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain (ATCC 25922GFP), the green fluorescent protein (GFP) gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth (TSB) (Becton, Dickinson and Company, Sparks, MD) containing 100 g/ml ampicillin (ATCC medium 2855). The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm (Bemis, Inc., Oshkosh, WI) and allowed to incubate at room temperature (22 Ϯ 2°C) for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar (Becton, Dickinson and Company, Sparks, MD) plates containing 100 g/ml ampicillin (TSA) to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks (Fig. 5a) . The remainder of the drain line was either autoclaved (strainer, tailpipe, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI), maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution (saline) were added in the ratio 1:3 via the strainer (Fig. 5b) to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece (between the P-trap and the strainer) and the trap arm (between the P-trap and the common line). These holes were fitted with size 00 silicone stoppers (Cole-Parmer, Vernon Hills, IL) (Fig. 5a) . Sterile cotton swabs (Covidien, Mansfield, MA) presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface (ϳ20 cm 2 ) of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink (sink 5) (Fig. 4) was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml (colonized for 14 days)-were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization (MALDI)-time of flight (MALDI-TOF) mass spectrometer (Vitek-MS; bioMérieux, Durham, NC). The wastewater paths of sinks 1 to 4 were either autoclaved (strainer, tailpipe, P-traps, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI). Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres (Polysciences, Inc.) which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres (ϳ10 10 particles) was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks (American Standard, model 7723018.002) (Fig. 5c) . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes (Covidien Webcol 6818; Kendall), and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position (Fig. 6a) . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system (Bio-Rad Laboratories, Inc.) with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software (Bio-Rad Laboratories, Inc.). To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension (ϳ10 10 particles/ml) using a disposable swab (Sage Products, Inc., Cary, IL), and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved (strainer, tailpipe, and trap arms). Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece (Fig. 5c ). Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli (for 14 days) was installed, and a nutrient regimen (Fig. 5b) was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform (Fig. 6b) . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area (CFU per square centimeter) was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate.

Which type of bacteria are implicated in carrying genes of drug resistance?

Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377511/ SHA: 615071c8c959f24857b1bad521cc432b59719bfb Authors: Kotay, Shireen; Chai, Weidong; Guilford, William; Barry, Katie; Mathers, Amy J. Date: 2017-03-31 DOI: 10.1128/aem.03327-16 License: cc-by Abstract: There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports (1) (2) (3) (4) (5) , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link (6) (7) (8) (9) (10) (11) (12) (13) . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents (14, 15) . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap (6, (16) (17) (18) (19) (20) . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities (16, 21, 22) . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed (23) (24) (25) (26) (27) (28) . There are many reports of a genetic association between pathogens found in sink traps and those found in patients (29, 30) . However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles (Glo Germ; Glo Germ Co., Moab, UT) that material injected into the P-trap gets dispersed around a hand-washing sink (6) . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: (i) can organisms grow retrograde from the P-trap water to the sink strainer, (ii) can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and (iii) which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing (30) . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein (GFP)-expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth (TSB) promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap (Table 1) . On day 7, the strainer (ϳ8 in. above the liquid in the P-trap) was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink (sink 5) was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration (10 3 CFU/ml) in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps (Fig. 1a) . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days (Fig. 1b and c) . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen (described previously) was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space (Fig. 2) . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl (zones 2, 5, 6, 9, 11, and 12) . Anterior corners of the sink counter space (zones 4 and 7), which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres (Fig. 2) . Dispersion was significantly higher along the axes (zones 6, 9, 11, and 12) and lower at the corners of the sink counter space (zones 4, 7, and 10). In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl (Fig. 3b) . Further, dispersion was greater along the Ϫ" and "ϩ" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl (Fig. 3b , zones B3, B4, and B10) than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets (Fig. 3a , zones 12 and 11). A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space (Fig. 3a , zones 4, 7, and 10) was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface (Fig. 3c ). There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done (6, 28) . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission (29, 31, 32) . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms (3, 33) . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles (6) . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported (6) . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks (intravenous fluids, feeding supplements, and leftover beverages) (5, 32) . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing (23) . Sink 3 was lowest on the slope in the drain line (see Fig. 4 ) with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source (sink 5), and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome (SARS) and Ebola viruses through premise plumbing systems (34) (35) (36) . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies (7, 10, 13, 24, 37) was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water (17, 20) . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal (6) . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users (38) . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other (Fig. 4) . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet (Elkay, Oak Brook, IL). The drain line (Dearborn Brass-Oatey, Cleveland, OH). All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 (Fig. 4) . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain (ATCC 25922GFP), the green fluorescent protein (GFP) gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth (TSB) (Becton, Dickinson and Company, Sparks, MD) containing 100 g/ml ampicillin (ATCC medium 2855). The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm (Bemis, Inc., Oshkosh, WI) and allowed to incubate at room temperature (22 Ϯ 2°C) for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar (Becton, Dickinson and Company, Sparks, MD) plates containing 100 g/ml ampicillin (TSA) to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks (Fig. 5a) . The remainder of the drain line was either autoclaved (strainer, tailpipe, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI), maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution (saline) were added in the ratio 1:3 via the strainer (Fig. 5b) to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece (between the P-trap and the strainer) and the trap arm (between the P-trap and the common line). These holes were fitted with size 00 silicone stoppers (Cole-Parmer, Vernon Hills, IL) (Fig. 5a) . Sterile cotton swabs (Covidien, Mansfield, MA) presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface (ϳ20 cm 2 ) of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink (sink 5) (Fig. 4) was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml (colonized for 14 days)-were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization (MALDI)-time of flight (MALDI-TOF) mass spectrometer (Vitek-MS; bioMérieux, Durham, NC). The wastewater paths of sinks 1 to 4 were either autoclaved (strainer, tailpipe, P-traps, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI). Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres (Polysciences, Inc.) which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres (ϳ10 10 particles) was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks (American Standard, model 7723018.002) (Fig. 5c) . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes (Covidien Webcol 6818; Kendall), and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position (Fig. 6a) . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system (Bio-Rad Laboratories, Inc.) with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software (Bio-Rad Laboratories, Inc.). To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension (ϳ10 10 particles/ml) using a disposable swab (Sage Products, Inc., Cary, IL), and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved (strainer, tailpipe, and trap arms). Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece (Fig. 5c ). Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli (for 14 days) was installed, and a nutrient regimen (Fig. 5b) was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform (Fig. 6b) . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area (CFU per square centimeter) was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate.

What may be a likely cause of sink-to-sink spreading of pathogens in the hospital setting?

The impact of rapid molecular diagnostic testing for respiratory viruses on outcomes for emergency department patients https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617970/ SHA: eea9d5e3d2244b3ecfb5e909515e00a4a3cabaa7 Authors: Wabe, Nasir; Li, Ling; Lindeman, Robert; Yimsung, Ruth; Dahm, Maria R; Clezy, Kate; McLennan, Susan; Westbrook, Johanna; Georgiou, Andrew Date: 2019-03-05 DOI: 10.5694/mja2.50049 License: cc-by Abstract: OBJECTIVE: To determine whether rapid polymerase chain reaction (PCR) testing for influenza and respiratory syncytial viruses (RSV) in emergency departments (EDs) is associated with better patient and laboratory outcomes than standard multiplex PCR testing. DESIGN, SETTING: A before‐and‐after study in four metropolitan EDs in New South Wales. PARTICIPANTS: 1491 consecutive patients tested by standard multiplex PCR during July–December 2016, and 2250 tested by rapid PCR during July–December 2017. MAIN OUTCOME MEASURES: Hospital admissions; ED length of stay (LOS); test turnaround time; patient receiving test result before leaving the ED; ordering of other laboratory tests. RESULTS: Compared with those tested by standard PCR, fewer patients tested by rapid PCR were admitted to hospital (73.3% v 77.7%; P < 0.001) and more received their test results before leaving the ED (67.4% v 1.3%; P < 0.001); the median test turnaround time was also shorter (2.4 h [IQR, 1.6–3.9 h] v 26.7 h [IQR, 21.2–37.8 h]). The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 (50.6% v 66.6%; P < 0.001) and patients over 60 years of age (84.3% v 91.8%; P < 0.001). Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid PCR. ED LOS was similar for the rapid (7.4 h; IQR, 5.0–12.9 h) and standard PCR groups (6.5 h; IQR, 4.2–11.9 h; P = 0.27). CONCLUSION: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost–benefit analysis should be undertaken. Text: The health and economic burdens associated with acute respiratory infections by influenza and respiratory syncytial viruses (RSV) are significant in Australia and overseas. 1-3 Polymerase chain reaction (PCR) testing is effective for confirming respiratory viral infections. 4 Multiplex PCR can detect numerous respiratory viruses, including influenza and parainfluenza viruses, RSV, adenovirus, rhinovirus, human metapneumovirus, enterovirus, bocavirus and coronavirus with very high sensitivity and specificity. 5 Although the results of standard multiplex PCR are accurate and comprehensive, it has traditionally been performed in a central laboratory with a lengthy turnaround time, which may be inconvenient in settings where test results are urgently required, including emergency departments (EDs). Rapid, easy-to-use PCR-based respiratory virus diagnostic tests have been introduced in recent years; 6,7 the GeneXpert system (Cepheid), for instance, was introduced in New South Wales in July 2017. Rapid PCR tests were expected to facilitate timely and appropriate initiation of treatment, improve outbreak prevention and infection control measures, and expedite the assessment of patients in EDs. In this study, we analysed routinely collected data to determine whether rapid PCR testing for influenza and RSV infections in EDs is associated with improved patient and laboratory outcomes. We compared data for patients tested for influenza A/B viruses and RSV immediately after rapid PCR diagnosis was introduced (July-December 2017) with data for patients tested with a standard multiplex PCR system during July-December 2016. We undertook a before-and-after study in four metropolitan public hospital EDs in Sydney, NSW: three general hospitals (EDs A, B and C; 76 228, 54 443 and 50 025 annual ED presentations respectively) and one children's hospital (ED D; 36 700 annual ED presentations; all data for January-December 2016 8 ). The four hospitals were served by a single pathology laboratory provider. We analysed data for all patients tested for influenza virus or RSV. During July-December 2016, patients were tested with the standard PCR system, a central laboratory-based multiplex PCR test for sixteen respiratory viruses (including RSV and influenza viruses A and B), available as a referral test at the central laboratory in Hospital B. During July-December 2017, patients were tested with the rapid PCR system, a hospital laboratory-based test specific for RSV and influenza viruses A and B. Hospitals A, B and D have onsite laboratories that perform rapid PCR testing; Hospital C sends samples to the nearby Hospital A. All tests were conducted in virology laboratories by trained staff, and test results were entered into laboratory information system datasets. We obtained relevant patient characteristics and The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 (50.6% v 66.6%; P < 0.001) and patients over 60 years of age (84.3% v 91.8%; P < 0.001). Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid PCR. ED LOS was similar for the rapid (7.4 h; IQR, 5.0-12.9 h) and standard PCR groups (6.5 h; IQR, 4.2-11.9 h; P = 0.27). Conclusion: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost-benefit analysis should be undertaken. The known: Rapid polymerase chain reaction (PCR) testing for influenza and respiratory syncytial viruses (RSV) was introduced in New South Wales in July 2017. Its impact on outcomes for emergency department (ED) patients has not been investigated. The new: Compared with standard PCR testing, rapid PCR was associated with significantly fewer hospital admissions, more rapid test turnaround, more patients receiving test results before leaving the ED, and reduced numbers of some other common microbiology tests. It did not significantly affect ED length of stay. The implications: Rapid PCR testing of ED patients for major respiratory viruses can benefit patients and reduce resource use. MJA 210 (7) ▪ 15 April 2019 317 laboratory data by linking the ED and laboratory information system datasets. Detailed information about the datasets and the linkage process have been described previously. 9 The primary outcomes were admission to hospital, ED length of stay (LOS), test turnaround time, and the patient receiving their test result before leaving the ED. ED LOS was defined as the time from arrival in the ED to discharge or admission to hospital. Turnaround was defined as the time from when the sample was received by the laboratory to when the test result was available in hospital electronic records. As secondary outcomes, we compared ordering of typical biochemistry and haematology tests (full blood count; electrolyte, urea, creatinine levels; liver function test; blood gas analysis; C-reactive protein level) and microbiology tests (blood culture; urine microscopy, culture and sensitivity analysis; sputum culture; respiratory bacteria and virus serology) during the two study periods. Analyses were conducted in Stata 15 (StataCorp). Descriptive statistics are reported (medians with interquartile ranges [IQRs], means with standard deviations [SDs], numbers with proportions). Baseline characteristics were compared in χ 2 tests (categorical outcomes) or Wilcoxon rank-sum tests (continuous outcomes). Logistic regression analyses of binary outcomes (eg, hospital admission: yes/no) and quantile regression analyses of continuous outcomes (eg, ED LOS) were undertaken. All analyses were adjusted for baseline characteristics. Sensitivity analyses limited to data for children (under 18 years of age) or older patients (over 60 years of age) were conducted. P < 0.05 (2-tailed) was deemed statistically significant. Ethics approval for the investigation was granted by the Human Research Ethics Committee of the South Eastern Sydney Local Health District (reference, HREC/16/POWH/412). We analysed data for 3741 patients presenting to the four EDs during two periods: 1491 consecutive patients during July-December 2016 (standard PCR) and 2250 during July-December 2017 (rapid PCR). Baseline characteristics for the two groups were similar in terms of sex, triage category, and arrival day of the week, but differed significantly for age, arrival time, and mode of arrival (Box 1 The overall numbers of tests per patient were similar in the standard PCR (mean, 7.2 tests; SD, 3.8) and rapid PCR groups (mean, 7.1 tests; SD, 3.4). The mean number of microbiology tests per patient was significantly lower for the rapid PCR group (1.5 tests; SD, 1.8) than for the standard PCR group (2.0 tests; SD, 2.1; P < 0.001 after controlling for baseline characteristics). The 16 265 biochemistry/haematology and microbiology tests comprised 71.1% of the 22 876 other tests (that is, not including PCR virus testing) ordered for patients in the study. After adjusting for baseline characteristics, the proportions of patients for whom full blood count, electrolyte/urea/creatinine levels, liver function, or C-reactive protein were assessed were similar, as were the proportions for urine microscopy, culture and sensitivity tests. Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients in the rapid PCR group (Box 4). Information, figure 1 ). ED LOS was similar for the standard PCR and rapid PCR groups in both age-based subgroups (Supporting Information, figure 2A ). The differences in test turnaround time identified in the main analysis were also evident for each age-based subgroup (Supporting Information, figure 2B ). In this before-and-after study, we found that rapid PCR testing of ED patients for major respiratory viruses was associated with significantly fewer admissions to hospital, more rapid delivery of test results, more patients receiving their test results before leaving the ED, and less frequent ordering of some common microbiology tests. Other studies have also reported that hospital admission numbers were significantly lower when rapid influenza virus testing was used in EDs. An analysis of outcomes for more than 300 adults at a tertiary care centre in New York found that early diagnosis of respiratory infections was associated with significantly fewer hospitalisations of influenza-positive patients. 7 In a small Irish study (73 patients), the hospital admission rate for obstetric patients declined from 88% to 45% after on-site rapid influenza PCR testing was introduced. 10 The differences in clinical setting and patient group may explain the smaller decline in our study (from 78% to 67%). Non-PCR-based rapid diagnostic tests for respiratory viruses have also been associated with lower hospital admission rates. 11, 12 The main reason for fewer hospital admissions of patients tested by rapid PCR may be that the earlier availability of results enables clinicians to quickly diagnose or exclude respiratory infections and to make timely and informed decisions about whether to discharge the patient or admit them to hospital. When standard 2 Primary outcomes for 3741 patients at four metropolitan emergency departments (EDs) tested for influenza and respiratory syncytial viruses by standard or rapid polymerase chain reaction (PCR) After adjusting for baseline characteristics (Box 1): * P = 0.012; ** P < 0.001. ◆ MJA 210 (7) ▪ 15 April 2019 PCR was used, in contrast, our findings suggest that these decisions were made before the test results were available. The possible benefits of not admitting patients to hospital, beyond those for individual patient management, include better infection control and outbreak prevention, as well as reduced demands on hospital resources. 13, 14 The impact of rapid PCR testing on outbreak prevention and infection control measures should be evaluated. Rapid influenza virus testing may also have practical implications for hospital bed management. 10, 15 ED LOS was similar in our study before and after the introduction of rapid PCR methods. This finding was not unexpected, as test turnaround time is not the only rate-limiting factor for decision making in EDs. 16 Before rapid PCR methods were introduced, the long turnaround time of multiplex PCR did not necessarily extend a patient's stay in the ED, as they were usually admitted to hospital or discharged home before the results were available. Consequently, more rapid delivery of test results alone would not reduce ED LOS. Reports on the effect of rapid influenza virus testing and LOS have been conflicting. While evidence for an association between rapid testing and shorter overall inpatient LOS has been reported, 6,11 findings regarding ED LOS are inconsistent. 7, 17, 18 For example, a Cochrane review based on three randomised controlled trials did not find a statistically significant association of rapid viral diagnosis with lower mean ED LOS. 18 In a study in children, ED LOS was actually 26 minutes longer with rapid PCR; inpatient LOS did not differ between the two groups, but was significantly shorter when the analysis was limited to patients with positive test results. 6 We found that ordering of some other microbiology tests, including blood culture, sputum culture, and respiratory bacterial and virus serology, was significantly less frequent for patients tested by rapid PCR. The effect of PCR-based rapid testing on the ordering of other laboratory tests has not previously been reported, although some studies of antigen-based pointof-care testing have examined this outcome. 12 Consistent with our finding, several investigators have reported fewer blood culture tests 19, 20 and basic biochemistry and haematology tests, including full blood count, 20,21 C-reactive protein testing, 21 and urinalysis, 20,21 when point-of-care testing was used. The higher rate of positive results for patients tested by rapid PCR than for those tested by standard PCR may reflect a higher prevalence of influenza during 2017 than in 2016. The rapid PCR system in our study accurately detects influenza viruses A/B and RSV but, unlike the standard multiplex PCR, cannot detect other clinically relevant respiratory viruses, such as rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, adenovirus, enterovirus, and bocavirus. If infection with other respiratory viruses is suspected, patients may therefore need further investigations. Standard multiplex PCR can provide broader information, as it can detect multiple respiratory viruses in a single run, although the long turnaround time restricts its suitability for urgent clinical decision making. Improving the turnaround time of multiplex PCR analysis may achieve better outcomes. The strengths of our study include its relatively large sample size; further, unlike many similar investigations, ours was a multicentre study in four hospital EDs, enhancing the generalisability of our findings. However, our analyses were not adjusted for comorbid conditions, as this information was not available. Because our study was an uncontrolled before-and-after study, our results cannot be interpreted as indicating causal relationships. As with all non-randomised trials, we could not fully account for all potential confounding variables, nor for temporal trends and other unmeasured factors, such as changes in clinician testing practices or differences in the prevalence and severity of disease during the two study periods. 22 For example, a shortage of inpatient beds caused by a high prevalence of influenza could influence decisions in a busy ED about admitting patients to hospital. However, we attempted to reduce seasonal effects by selecting similar time frames for the two study periods, to reduce selection bias by including all ED patients tested for influenza virus and RSV, and to control for differences in baseline patient characteristics by applying multivariate modelling. As medications data were not available to us, we were unable to assess the impact of rapid PCR testing on antibiotic and antiviral drug use. Similarly, the cost-benefit balance of rapid testing was not evaluated because relevant data were not available. The cost per patient of rapid PCR testing is generally higher than for central laboratory testing, but our findings suggest potential savings through lower numbers of hospital admissions and reduced resource use. This question could be evaluated in a further study. Rapid PCR testing for influenza virus and RSV infections in patients attending EDs was associated with significant improvements in a range of patient and laboratory outcomes, suggesting potential benefits for both the patients and the health care system. A cost-benefit analysis could examine the impact of rapid PCR testing on bed management and antimicrobial drug prescribing.

What types of acute respiratory infections can be screened and diagnosed with multiplex PCR?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

What is the role of antibodies during infection?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

How can antibodies also create health problems?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

Which technology invention produced antibodies that are clones of a unique parent cell?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

What mechanism is responsible for the creation of diversified repertoire for antibodies?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

What developments have been made possible by the study of B-cell repertoire?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

What motivates the study of the rare B-cells that produce Broadly Neutralizing Antibodies (bnAb)?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

How has the study of B-cells helped the treatment for Respiratory syncytial virus (RSV)?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

How are the studies on B-cells helping the development of a universal influenza vaccine?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

What role B-cell play in malaria infection and prevention?

Techniques to Study Antigen-Specific B Cell Responses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/ SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c Authors: Boonyaratanakornkit, Jim; Taylor, Justin J. Date: 2019-07-24 DOI: 10.3389/fimmu.2019.01694 License: cc-by Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31). Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities; (2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript.

How can the study of B-cells help in the prevention and treatment of autoimmune diseases?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

When was the Middle East Respiratory Syndrome Coronavirus isolated first?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

What is the Case fatality rate for MERS Coronavirus?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

How does gender influence MERS-COV infection?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

Which is the source animal for the MERS-COV?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

What is the median time until death in MERS-COV?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

What is the incubation period for MERS-COV?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

What is the treatment for MERS-COV?

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049846/ SHA: edee452881f826fb72c58ee68a982789b12aa99d Authors: Altamimi, Asmaa; Abu-Saris, Raghib; El-Metwally, Ashraf; Alaifan, Taghreed; Alamri, Aref Date: 2020-02-19 DOI: 10.1155/2020/9629747 License: cc-by Abstract: Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus (MERS-CoV) was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure [1] . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea [2] . Recent fatality rate (CFR) of 21% [5, 6] . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that MERS-CoV infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings [4] . Studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays [14] . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic [2] . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness [4] . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation [16] . Another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific [18, 19] . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution (CBAHI) 2018 [21] . Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection [20] . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period (2017). A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN (health electronic surveillance networks) and to the World Health Organization (WHO) through the International Health Regulations (IHR), National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in KSA (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO (children: 0-14, adult: otherwise) [20] . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in WHO reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 [14] . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above [24] . In 2016, only 9 of 552 cases (1.6%) of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh (KFMC) between January 2012 and December 2013 did not report any MERS-CoV cases among children [26] . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above [14] . Similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . Similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (AST) [21] . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas (two major regions in KSA). Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests.

What age group had the most MERS-COV infections?

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923505/ SHA: f1e1e2511e051195c8327a56d5c311a2dd4ab6b3 Authors: Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan Date: 2018-04-20 DOI: 10.3390/v10040211 License: cc-by Abstract: Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases [1] . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (HIV), and sofosbuvir for hepatitis C virus (HCV)) have been successful in clinical trials [2] [3] [4] [5] and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α [6] . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV (EC 50 of 1.2 µM and 4.9 µM, respectively) [13] . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium (RPE) cells, particularly at non-cytotoxic concentrations (EC 50 of 0.01 µM vs. CC 50 of > 10 µM) [14] . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively [17, 19] , which were lower concentrations than those used in cancer therapy [20] . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency [21] . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus (MuLV), in vitro (EC 50 of 1.6 nM) and even in murine AIDS model [17] . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. (EC 50 of 0.068 µM) [16] . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 (HSV-1) (>2 log reduction in virus titer) but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus (MeV), and vaccinia virus [16] . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 (CVB3), was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group (EC 50 of 0.4 µM) [18] . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 (EV71) and human rhinoviruses (HRVs) (EC 50 s of 1 and 1-5 µM, respectively). In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [22] . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells (EC 50 of 0.3 µM) [15] . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine [16] . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy [14, 20] . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 [18] . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo [17, 21] . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [17, 22] . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis [23] . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells [16] . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 [6, 10, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (MPA) are inhibitors of inosine-5 -monophosphate (IMP) dehydrogenase (IMPDH), which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus (HEV), MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus (WNV), Chikungunya virus (CHIKV), and IAV [24] [25] [26] [27] [28] [29] [30] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs [10, 30] . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses [6] . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine [10] . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin [30] . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized [10] . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element (ISRE)-stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV [37] . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 (IRF1) transcription factor, a master regulator of antiviral gene expression [37] , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 [30] . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 [38] . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal [6, 10, 23] . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK [10] . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions [6] . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine [23] . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation [43, 44] . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the (d)NTP pool. Inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 (IFN-stimulated gene factor 3; ISGF3), at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin [6] . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns [10] . Targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses [45] . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.

Why are nucleosides analogs used for chemotheraphy?

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923505/ SHA: f1e1e2511e051195c8327a56d5c311a2dd4ab6b3 Authors: Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan Date: 2018-04-20 DOI: 10.3390/v10040211 License: cc-by Abstract: Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases [1] . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (HIV), and sofosbuvir for hepatitis C virus (HCV)) have been successful in clinical trials [2] [3] [4] [5] and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α [6] . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV (EC 50 of 1.2 µM and 4.9 µM, respectively) [13] . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium (RPE) cells, particularly at non-cytotoxic concentrations (EC 50 of 0.01 µM vs. CC 50 of > 10 µM) [14] . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively [17, 19] , which were lower concentrations than those used in cancer therapy [20] . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency [21] . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus (MuLV), in vitro (EC 50 of 1.6 nM) and even in murine AIDS model [17] . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. (EC 50 of 0.068 µM) [16] . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 (HSV-1) (>2 log reduction in virus titer) but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus (MeV), and vaccinia virus [16] . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 (CVB3), was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group (EC 50 of 0.4 µM) [18] . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 (EV71) and human rhinoviruses (HRVs) (EC 50 s of 1 and 1-5 µM, respectively). In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [22] . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells (EC 50 of 0.3 µM) [15] . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine [16] . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy [14, 20] . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 [18] . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo [17, 21] . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [17, 22] . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis [23] . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells [16] . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 [6, 10, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (MPA) are inhibitors of inosine-5 -monophosphate (IMP) dehydrogenase (IMPDH), which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus (HEV), MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus (WNV), Chikungunya virus (CHIKV), and IAV [24] [25] [26] [27] [28] [29] [30] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs [10, 30] . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses [6] . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine [10] . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin [30] . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized [10] . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element (ISRE)-stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV [37] . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 (IRF1) transcription factor, a master regulator of antiviral gene expression [37] , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 [30] . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 [38] . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal [6, 10, 23] . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK [10] . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions [6] . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine [23] . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation [43, 44] . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the (d)NTP pool. Inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 (IFN-stimulated gene factor 3; ISGF3), at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin [6] . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns [10] . Targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses [45] . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.

What nucleoside analog is the focus of the current study?

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923505/ SHA: f1e1e2511e051195c8327a56d5c311a2dd4ab6b3 Authors: Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan Date: 2018-04-20 DOI: 10.3390/v10040211 License: cc-by Abstract: Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases [1] . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (HIV), and sofosbuvir for hepatitis C virus (HCV)) have been successful in clinical trials [2] [3] [4] [5] and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α [6] . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV (EC 50 of 1.2 µM and 4.9 µM, respectively) [13] . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium (RPE) cells, particularly at non-cytotoxic concentrations (EC 50 of 0.01 µM vs. CC 50 of > 10 µM) [14] . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively [17, 19] , which were lower concentrations than those used in cancer therapy [20] . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency [21] . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus (MuLV), in vitro (EC 50 of 1.6 nM) and even in murine AIDS model [17] . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. (EC 50 of 0.068 µM) [16] . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 (HSV-1) (>2 log reduction in virus titer) but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus (MeV), and vaccinia virus [16] . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 (CVB3), was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group (EC 50 of 0.4 µM) [18] . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 (EV71) and human rhinoviruses (HRVs) (EC 50 s of 1 and 1-5 µM, respectively). In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [22] . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells (EC 50 of 0.3 µM) [15] . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine [16] . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy [14, 20] . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 [18] . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo [17, 21] . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [17, 22] . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis [23] . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells [16] . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 [6, 10, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (MPA) are inhibitors of inosine-5 -monophosphate (IMP) dehydrogenase (IMPDH), which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus (HEV), MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus (WNV), Chikungunya virus (CHIKV), and IAV [24] [25] [26] [27] [28] [29] [30] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs [10, 30] . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses [6] . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine [10] . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin [30] . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized [10] . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element (ISRE)-stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV [37] . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 (IRF1) transcription factor, a master regulator of antiviral gene expression [37] , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 [30] . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 [38] . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal [6, 10, 23] . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK [10] . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions [6] . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine [23] . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation [43, 44] . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the (d)NTP pool. Inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 (IFN-stimulated gene factor 3; ISGF3), at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin [6] . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns [10] . Targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses [45] . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.

Gemcitabine has been shown to have antiviral activity against which viruses?

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923505/ SHA: f1e1e2511e051195c8327a56d5c311a2dd4ab6b3 Authors: Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan Date: 2018-04-20 DOI: 10.3390/v10040211 License: cc-by Abstract: Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases [1] . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (HIV), and sofosbuvir for hepatitis C virus (HCV)) have been successful in clinical trials [2] [3] [4] [5] and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α [6] . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV (EC 50 of 1.2 µM and 4.9 µM, respectively) [13] . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium (RPE) cells, particularly at non-cytotoxic concentrations (EC 50 of 0.01 µM vs. CC 50 of > 10 µM) [14] . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively [17, 19] , which were lower concentrations than those used in cancer therapy [20] . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency [21] . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus (MuLV), in vitro (EC 50 of 1.6 nM) and even in murine AIDS model [17] . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. (EC 50 of 0.068 µM) [16] . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 (HSV-1) (>2 log reduction in virus titer) but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus (MeV), and vaccinia virus [16] . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 (CVB3), was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group (EC 50 of 0.4 µM) [18] . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 (EV71) and human rhinoviruses (HRVs) (EC 50 s of 1 and 1-5 µM, respectively). In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [22] . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells (EC 50 of 0.3 µM) [15] . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine [16] . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy [14, 20] . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 [18] . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo [17, 21] . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [17, 22] . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis [23] . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells [16] . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 [6, 10, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (MPA) are inhibitors of inosine-5 -monophosphate (IMP) dehydrogenase (IMPDH), which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus (HEV), MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus (WNV), Chikungunya virus (CHIKV), and IAV [24] [25] [26] [27] [28] [29] [30] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs [10, 30] . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses [6] . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine [10] . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin [30] . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized [10] . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element (ISRE)-stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV [37] . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 (IRF1) transcription factor, a master regulator of antiviral gene expression [37] , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 [30] . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 [38] . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal [6, 10, 23] . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK [10] . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions [6] . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine [23] . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation [43, 44] . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the (d)NTP pool. Inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 (IFN-stimulated gene factor 3; ISGF3), at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin [6] . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns [10] . Targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses [45] . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.

How does gemcitabine disrupt viral activity?

Identification and characterisation of the CD40-ligand of Sigmodon hispidus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063397/ SHA: edf2997357501734a93c1b7e16d44e86a7d20853 Authors: Russell, Marsha S.; Muralidharan, Abenaya; Larocque, Louise; Cao, Jingxin; Deschambault, Yvon; Varga, Jessie; Thulasi Raman, Sathya N.; Li, Xuguang Date: 2018-07-27 DOI: 10.1371/journal.pone.0199067 License: cc-by Abstract: Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1] , and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4] . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles (paramyxovirus) [5] , herpes simplex (oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic) [6] , influenza (orthomyxovirus) [7, 8] , HIV-1 [9] , RSV (respiratory syncytial virus) [10] , adenovirus [11, 12] , human parainfluenza [13] , and human metapneumovirus [14] . This model has been valuable for adenovirus-based gene replacement therapy research [15, 16] , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies (RespiGam 1 [17] , and Synagis 1 [18] . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials [19, 20] . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand (CD40L), remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells [21, 22] . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands [23] . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells (DCs) [24] , B cells [25] , and platelets [26] . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells [28] .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes [29, 30] . Here we report the successful cloning of the gene encoding cotton rat CD40L (crCD40L); we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. [6] [7] weeks old cotton rats were obtained from an inbred colony maintained at Envigo (USA). All animal experiments were conducted in accordance with Institutional Care and Use Committee (IACUC) of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II (Qiagen) and total RNA extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion according to the user's manual. The 3' RACE system (Life Technologies) was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer (5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3') was derived from a consensus sequence aligning the rat (Rattus norvegicus UniProt: Q9Z2V2), mouse (Mus musculus UniProt: P27548), and golden hamster (Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3) CD40L sequences obtained from the National Center for Biotechnology Information (NCBI). Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer (5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3') to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit (ThermoFisher cat # 4336917). Briefly, samples were amplified in a PTC-200 thermal cycle (MJ Research) with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit (Qiagen cat # 63204) and loaded onto a 3130xl Genetic Analyzer (Applied Biosystems). Raw sequencing data was edited by the instrument's software (ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0), and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast. cgi) to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST (blastp algorithm; https://blast.ncbi.nlm.nih.gov). The sequences producing significant alignments were imported into Geneosis software, (Auckland, New Zealand). Multiple alignment was conducted as previously described [31] , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence (5'-CACCGCCGCCACC-3'), followed by a secretion signal consisting of 23 amino acid (aa) (MLLAVLYCLLWSFQTSAGHFPRA) from the human tyrosinase signal peptide as previously described [32] . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added [33] and finally connected to the full length 783bp open reading frame (ORF) of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 (Biobasic, Markham, ON). Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker (Jingxin Cao, unpublished information). Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene (SOD homolog); the CD40L construct gene driven by a modified vaccinia H5 promoter (Vaccine 1996, 14:1451), and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns (Qiagen). Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer (Bio-Rad Laboratories Inc.), and the protein samples were transferred to Immobilon-FL PVDF membranes (Millipore). Protein was detected with Tetra-HIS Ab (Qiagen) and goat anti-mouse IRDye-800CW (LiCor). Membranes were developed using the Odyssey system (LiCor). The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer (10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0) and disrupted by sonication on ice using a Branson sonifier 150 (ThermoFisher, Waltham, MA) at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin (Qiagen, Mississauga, ON, Canada) (10 mL resin bed) and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier (Amersham Biosciences) with Unicorn 5.3 software (Amersham Biosciences). Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B (buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8) over 10 column volumes (CVs). The column was then washed with three CVs of buffer B + 60 mM imidazole (pH 7.8) to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole (pH 7.8). The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L (R&D Systems) or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer (PBS-0.1% tween-20) and then blocked with 200μl/well blocking buffer (PBS containing 0.1% Tween 20 and 3%IgG Free BSA) for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L (R&D Systems) 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate (Zymed). Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate (New England Bio Labs). The reaction was stopped with Stop solution (New England Bio Labs) and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice (Chicago, IL) were thawed and cultured in dendritic cell medium from manufacture (Cell Biologics M7711) supplemented with GMCSF (Cell Biologics) without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L (Preprotech, Montreal, QC) or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system (Applied Biosystems). TaqMan assay reagent kits (Applied Biosystems) were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit (Qiagen) according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix (Applied Biosystems) according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification [34] , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit (R & D Systems) following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps (Fig 1) . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid (aa) level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii (or deer mouse) at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor (TNF) superfamily Using EZmol software [35] , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family (hamster and deer mouse) than to those of the Muridae family (rat and mouse) as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites [36] . TNF superfamily members include TNF (TNF-alpha), LT (lymphotoxin-alpha, TNF-beta), CD40 ligand, Apo2L (TRAIL), Fas ligand, and osteoprotegerin (OPG) ligand, among others [37] . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB [37] . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues [23] . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains [38] . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding [23, 39] . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain [23, 40] , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain [23, 40] , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif (Fig 4a) . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody (Ab) was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant (faint band-48 hours only). Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate (data not shown). This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification [36] . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration [23] . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations ( Fig 5) . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea [41] . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 (n = 2). Data shown is a representative experiment of three separate experiments where two (n = 2) technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. https://doi.org/10.1371/journal.pone.0199067.g005 addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function [36] , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions [42] . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated [43] . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L [44, 45] . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L [45] . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used (Tables 1 and 2 ). CD11c was down regulated in both median flouresence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity (Table 1) but not up-regulated in terms of the overall percentage of positive cells (Table 2) . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses [46] . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival [47] [48] [49] [50] . We tested if the recombinant crCD40L could induce IL-6 gene expression (Fig 6a) and production of the cytokine (Fig 6b) by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers (CD40, CD54, CD80, and CD86) as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses [51, 52] .

Why are cotton rats considered a strong animal model for biomedical research?

Identification and characterisation of the CD40-ligand of Sigmodon hispidus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063397/ SHA: edf2997357501734a93c1b7e16d44e86a7d20853 Authors: Russell, Marsha S.; Muralidharan, Abenaya; Larocque, Louise; Cao, Jingxin; Deschambault, Yvon; Varga, Jessie; Thulasi Raman, Sathya N.; Li, Xuguang Date: 2018-07-27 DOI: 10.1371/journal.pone.0199067 License: cc-by Abstract: Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1] , and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4] . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles (paramyxovirus) [5] , herpes simplex (oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic) [6] , influenza (orthomyxovirus) [7, 8] , HIV-1 [9] , RSV (respiratory syncytial virus) [10] , adenovirus [11, 12] , human parainfluenza [13] , and human metapneumovirus [14] . This model has been valuable for adenovirus-based gene replacement therapy research [15, 16] , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies (RespiGam 1 [17] , and Synagis 1 [18] . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials [19, 20] . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand (CD40L), remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells [21, 22] . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands [23] . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells (DCs) [24] , B cells [25] , and platelets [26] . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells [28] .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes [29, 30] . Here we report the successful cloning of the gene encoding cotton rat CD40L (crCD40L); we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. [6] [7] weeks old cotton rats were obtained from an inbred colony maintained at Envigo (USA). All animal experiments were conducted in accordance with Institutional Care and Use Committee (IACUC) of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II (Qiagen) and total RNA extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion according to the user's manual. The 3' RACE system (Life Technologies) was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer (5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3') was derived from a consensus sequence aligning the rat (Rattus norvegicus UniProt: Q9Z2V2), mouse (Mus musculus UniProt: P27548), and golden hamster (Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3) CD40L sequences obtained from the National Center for Biotechnology Information (NCBI). Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer (5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3') to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit (ThermoFisher cat # 4336917). Briefly, samples were amplified in a PTC-200 thermal cycle (MJ Research) with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit (Qiagen cat # 63204) and loaded onto a 3130xl Genetic Analyzer (Applied Biosystems). Raw sequencing data was edited by the instrument's software (ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0), and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast. cgi) to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST (blastp algorithm; https://blast.ncbi.nlm.nih.gov). The sequences producing significant alignments were imported into Geneosis software, (Auckland, New Zealand). Multiple alignment was conducted as previously described [31] , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence (5'-CACCGCCGCCACC-3'), followed by a secretion signal consisting of 23 amino acid (aa) (MLLAVLYCLLWSFQTSAGHFPRA) from the human tyrosinase signal peptide as previously described [32] . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added [33] and finally connected to the full length 783bp open reading frame (ORF) of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 (Biobasic, Markham, ON). Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker (Jingxin Cao, unpublished information). Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene (SOD homolog); the CD40L construct gene driven by a modified vaccinia H5 promoter (Vaccine 1996, 14:1451), and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns (Qiagen). Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer (Bio-Rad Laboratories Inc.), and the protein samples were transferred to Immobilon-FL PVDF membranes (Millipore). Protein was detected with Tetra-HIS Ab (Qiagen) and goat anti-mouse IRDye-800CW (LiCor). Membranes were developed using the Odyssey system (LiCor). The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer (10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0) and disrupted by sonication on ice using a Branson sonifier 150 (ThermoFisher, Waltham, MA) at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin (Qiagen, Mississauga, ON, Canada) (10 mL resin bed) and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier (Amersham Biosciences) with Unicorn 5.3 software (Amersham Biosciences). Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B (buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8) over 10 column volumes (CVs). The column was then washed with three CVs of buffer B + 60 mM imidazole (pH 7.8) to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole (pH 7.8). The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L (R&D Systems) or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer (PBS-0.1% tween-20) and then blocked with 200μl/well blocking buffer (PBS containing 0.1% Tween 20 and 3%IgG Free BSA) for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L (R&D Systems) 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate (Zymed). Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate (New England Bio Labs). The reaction was stopped with Stop solution (New England Bio Labs) and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice (Chicago, IL) were thawed and cultured in dendritic cell medium from manufacture (Cell Biologics M7711) supplemented with GMCSF (Cell Biologics) without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L (Preprotech, Montreal, QC) or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system (Applied Biosystems). TaqMan assay reagent kits (Applied Biosystems) were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit (Qiagen) according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix (Applied Biosystems) according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification [34] , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit (R & D Systems) following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps (Fig 1) . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid (aa) level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii (or deer mouse) at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor (TNF) superfamily Using EZmol software [35] , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family (hamster and deer mouse) than to those of the Muridae family (rat and mouse) as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites [36] . TNF superfamily members include TNF (TNF-alpha), LT (lymphotoxin-alpha, TNF-beta), CD40 ligand, Apo2L (TRAIL), Fas ligand, and osteoprotegerin (OPG) ligand, among others [37] . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB [37] . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues [23] . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains [38] . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding [23, 39] . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain [23, 40] , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain [23, 40] , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif (Fig 4a) . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody (Ab) was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant (faint band-48 hours only). Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate (data not shown). This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification [36] . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration [23] . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations ( Fig 5) . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea [41] . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 (n = 2). Data shown is a representative experiment of three separate experiments where two (n = 2) technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. https://doi.org/10.1371/journal.pone.0199067.g005 addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function [36] , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions [42] . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated [43] . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L [44, 45] . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L [45] . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used (Tables 1 and 2 ). CD11c was down regulated in both median flouresence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity (Table 1) but not up-regulated in terms of the overall percentage of positive cells (Table 2) . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses [46] . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival [47] [48] [49] [50] . We tested if the recombinant crCD40L could induce IL-6 gene expression (Fig 6a) and production of the cytokine (Fig 6b) by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers (CD40, CD54, CD80, and CD86) as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses [51, 52] .

What is the structure of the CD40 Ligand?

Identification and characterisation of the CD40-ligand of Sigmodon hispidus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063397/ SHA: edf2997357501734a93c1b7e16d44e86a7d20853 Authors: Russell, Marsha S.; Muralidharan, Abenaya; Larocque, Louise; Cao, Jingxin; Deschambault, Yvon; Varga, Jessie; Thulasi Raman, Sathya N.; Li, Xuguang Date: 2018-07-27 DOI: 10.1371/journal.pone.0199067 License: cc-by Abstract: Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1] , and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4] . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles (paramyxovirus) [5] , herpes simplex (oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic) [6] , influenza (orthomyxovirus) [7, 8] , HIV-1 [9] , RSV (respiratory syncytial virus) [10] , adenovirus [11, 12] , human parainfluenza [13] , and human metapneumovirus [14] . This model has been valuable for adenovirus-based gene replacement therapy research [15, 16] , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies (RespiGam 1 [17] , and Synagis 1 [18] . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials [19, 20] . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand (CD40L), remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells [21, 22] . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands [23] . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells (DCs) [24] , B cells [25] , and platelets [26] . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells [28] .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes [29, 30] . Here we report the successful cloning of the gene encoding cotton rat CD40L (crCD40L); we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. [6] [7] weeks old cotton rats were obtained from an inbred colony maintained at Envigo (USA). All animal experiments were conducted in accordance with Institutional Care and Use Committee (IACUC) of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II (Qiagen) and total RNA extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion according to the user's manual. The 3' RACE system (Life Technologies) was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer (5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3') was derived from a consensus sequence aligning the rat (Rattus norvegicus UniProt: Q9Z2V2), mouse (Mus musculus UniProt: P27548), and golden hamster (Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3) CD40L sequences obtained from the National Center for Biotechnology Information (NCBI). Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer (5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3') to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit (ThermoFisher cat # 4336917). Briefly, samples were amplified in a PTC-200 thermal cycle (MJ Research) with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit (Qiagen cat # 63204) and loaded onto a 3130xl Genetic Analyzer (Applied Biosystems). Raw sequencing data was edited by the instrument's software (ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0), and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast. cgi) to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST (blastp algorithm; https://blast.ncbi.nlm.nih.gov). The sequences producing significant alignments were imported into Geneosis software, (Auckland, New Zealand). Multiple alignment was conducted as previously described [31] , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence (5'-CACCGCCGCCACC-3'), followed by a secretion signal consisting of 23 amino acid (aa) (MLLAVLYCLLWSFQTSAGHFPRA) from the human tyrosinase signal peptide as previously described [32] . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added [33] and finally connected to the full length 783bp open reading frame (ORF) of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 (Biobasic, Markham, ON). Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker (Jingxin Cao, unpublished information). Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene (SOD homolog); the CD40L construct gene driven by a modified vaccinia H5 promoter (Vaccine 1996, 14:1451), and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns (Qiagen). Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer (Bio-Rad Laboratories Inc.), and the protein samples were transferred to Immobilon-FL PVDF membranes (Millipore). Protein was detected with Tetra-HIS Ab (Qiagen) and goat anti-mouse IRDye-800CW (LiCor). Membranes were developed using the Odyssey system (LiCor). The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer (10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0) and disrupted by sonication on ice using a Branson sonifier 150 (ThermoFisher, Waltham, MA) at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin (Qiagen, Mississauga, ON, Canada) (10 mL resin bed) and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier (Amersham Biosciences) with Unicorn 5.3 software (Amersham Biosciences). Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B (buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8) over 10 column volumes (CVs). The column was then washed with three CVs of buffer B + 60 mM imidazole (pH 7.8) to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole (pH 7.8). The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L (R&D Systems) or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer (PBS-0.1% tween-20) and then blocked with 200μl/well blocking buffer (PBS containing 0.1% Tween 20 and 3%IgG Free BSA) for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L (R&D Systems) 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate (Zymed). Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate (New England Bio Labs). The reaction was stopped with Stop solution (New England Bio Labs) and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice (Chicago, IL) were thawed and cultured in dendritic cell medium from manufacture (Cell Biologics M7711) supplemented with GMCSF (Cell Biologics) without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L (Preprotech, Montreal, QC) or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system (Applied Biosystems). TaqMan assay reagent kits (Applied Biosystems) were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit (Qiagen) according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix (Applied Biosystems) according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification [34] , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit (R & D Systems) following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps (Fig 1) . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid (aa) level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii (or deer mouse) at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor (TNF) superfamily Using EZmol software [35] , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family (hamster and deer mouse) than to those of the Muridae family (rat and mouse) as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites [36] . TNF superfamily members include TNF (TNF-alpha), LT (lymphotoxin-alpha, TNF-beta), CD40 ligand, Apo2L (TRAIL), Fas ligand, and osteoprotegerin (OPG) ligand, among others [37] . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB [37] . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues [23] . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains [38] . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding [23, 39] . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain [23, 40] , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain [23, 40] , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif (Fig 4a) . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody (Ab) was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant (faint band-48 hours only). Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate (data not shown). This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification [36] . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration [23] . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations ( Fig 5) . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea [41] . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 (n = 2). Data shown is a representative experiment of three separate experiments where two (n = 2) technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. https://doi.org/10.1371/journal.pone.0199067.g005 addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function [36] , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions [42] . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated [43] . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L [44, 45] . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L [45] . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used (Tables 1 and 2 ). CD11c was down regulated in both median flouresence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity (Table 1) but not up-regulated in terms of the overall percentage of positive cells (Table 2) . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses [46] . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival [47] [48] [49] [50] . We tested if the recombinant crCD40L could induce IL-6 gene expression (Fig 6a) and production of the cytokine (Fig 6b) by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers (CD40, CD54, CD80, and CD86) as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses [51, 52] .

What is the effect of CD40L on Dendritic Cells?

Identification and characterisation of the CD40-ligand of Sigmodon hispidus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063397/ SHA: edf2997357501734a93c1b7e16d44e86a7d20853 Authors: Russell, Marsha S.; Muralidharan, Abenaya; Larocque, Louise; Cao, Jingxin; Deschambault, Yvon; Varga, Jessie; Thulasi Raman, Sathya N.; Li, Xuguang Date: 2018-07-27 DOI: 10.1371/journal.pone.0199067 License: cc-by Abstract: Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1] , and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4] . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles (paramyxovirus) [5] , herpes simplex (oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic) [6] , influenza (orthomyxovirus) [7, 8] , HIV-1 [9] , RSV (respiratory syncytial virus) [10] , adenovirus [11, 12] , human parainfluenza [13] , and human metapneumovirus [14] . This model has been valuable for adenovirus-based gene replacement therapy research [15, 16] , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies (RespiGam 1 [17] , and Synagis 1 [18] . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials [19, 20] . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand (CD40L), remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells [21, 22] . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands [23] . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells (DCs) [24] , B cells [25] , and platelets [26] . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells [28] .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes [29, 30] . Here we report the successful cloning of the gene encoding cotton rat CD40L (crCD40L); we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. [6] [7] weeks old cotton rats were obtained from an inbred colony maintained at Envigo (USA). All animal experiments were conducted in accordance with Institutional Care and Use Committee (IACUC) of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II (Qiagen) and total RNA extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion according to the user's manual. The 3' RACE system (Life Technologies) was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer (5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3') was derived from a consensus sequence aligning the rat (Rattus norvegicus UniProt: Q9Z2V2), mouse (Mus musculus UniProt: P27548), and golden hamster (Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3) CD40L sequences obtained from the National Center for Biotechnology Information (NCBI). Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer (5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3') to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit (ThermoFisher cat # 4336917). Briefly, samples were amplified in a PTC-200 thermal cycle (MJ Research) with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit (Qiagen cat # 63204) and loaded onto a 3130xl Genetic Analyzer (Applied Biosystems). Raw sequencing data was edited by the instrument's software (ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0), and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast. cgi) to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST (blastp algorithm; https://blast.ncbi.nlm.nih.gov). The sequences producing significant alignments were imported into Geneosis software, (Auckland, New Zealand). Multiple alignment was conducted as previously described [31] , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence (5'-CACCGCCGCCACC-3'), followed by a secretion signal consisting of 23 amino acid (aa) (MLLAVLYCLLWSFQTSAGHFPRA) from the human tyrosinase signal peptide as previously described [32] . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added [33] and finally connected to the full length 783bp open reading frame (ORF) of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 (Biobasic, Markham, ON). Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker (Jingxin Cao, unpublished information). Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene (SOD homolog); the CD40L construct gene driven by a modified vaccinia H5 promoter (Vaccine 1996, 14:1451), and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns (Qiagen). Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer (Bio-Rad Laboratories Inc.), and the protein samples were transferred to Immobilon-FL PVDF membranes (Millipore). Protein was detected with Tetra-HIS Ab (Qiagen) and goat anti-mouse IRDye-800CW (LiCor). Membranes were developed using the Odyssey system (LiCor). The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer (10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0) and disrupted by sonication on ice using a Branson sonifier 150 (ThermoFisher, Waltham, MA) at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin (Qiagen, Mississauga, ON, Canada) (10 mL resin bed) and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier (Amersham Biosciences) with Unicorn 5.3 software (Amersham Biosciences). Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B (buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8) over 10 column volumes (CVs). The column was then washed with three CVs of buffer B + 60 mM imidazole (pH 7.8) to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole (pH 7.8). The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L (R&D Systems) or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer (PBS-0.1% tween-20) and then blocked with 200μl/well blocking buffer (PBS containing 0.1% Tween 20 and 3%IgG Free BSA) for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L (R&D Systems) 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate (Zymed). Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate (New England Bio Labs). The reaction was stopped with Stop solution (New England Bio Labs) and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice (Chicago, IL) were thawed and cultured in dendritic cell medium from manufacture (Cell Biologics M7711) supplemented with GMCSF (Cell Biologics) without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L (Preprotech, Montreal, QC) or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system (Applied Biosystems). TaqMan assay reagent kits (Applied Biosystems) were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit (Qiagen) according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix (Applied Biosystems) according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification [34] , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit (R & D Systems) following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps (Fig 1) . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid (aa) level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii (or deer mouse) at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor (TNF) superfamily Using EZmol software [35] , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family (hamster and deer mouse) than to those of the Muridae family (rat and mouse) as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites [36] . TNF superfamily members include TNF (TNF-alpha), LT (lymphotoxin-alpha, TNF-beta), CD40 ligand, Apo2L (TRAIL), Fas ligand, and osteoprotegerin (OPG) ligand, among others [37] . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB [37] . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues [23] . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains [38] . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding [23, 39] . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain [23, 40] , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain [23, 40] , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif (Fig 4a) . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody (Ab) was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant (faint band-48 hours only). Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate (data not shown). This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification [36] . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration [23] . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations ( Fig 5) . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea [41] . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 (n = 2). Data shown is a representative experiment of three separate experiments where two (n = 2) technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. https://doi.org/10.1371/journal.pone.0199067.g005 addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function [36] , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions [42] . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells [27] . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated [43] . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L [44, 45] . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L [45] . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used (Tables 1 and 2 ). CD11c was down regulated in both median flouresence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity (Table 1 ) and the percentage of positive cells ( Table 2 ). The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity (Table 1) but not up-regulated in terms of the overall percentage of positive cells (Table 2) . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses [46] . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival [47] [48] [49] [50] . We tested if the recombinant crCD40L could induce IL-6 gene expression (Fig 6a) and production of the cytokine (Fig 6b) by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers (CD40, CD54, CD80, and CD86) as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses [51, 52] .

What is the effect of CD40L on B Cells?

Bioinformatics analysis of rabbit haemorrhagic disease virus genome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377956/ SHA: eff26d8739498efca2d32fe2e66cdbebf0569c50 Authors: Tian, Xiao-ting; Li, Bao-yu; Zhang, Liang; Jiao, Wen-qiang; Liu, Ji-xing Date: 2011-11-01 DOI: 10.1186/1743-422x-8-494 License: cc-by Abstract: BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly [1] . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors [2, 3] . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response [4] . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition [5] . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus [6] . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets [4] . Analysis of the bovine papillomavirus type 1 (BPV1) late genes has revealed a relationship between codon usage and tRNA availability [7] . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression [8] . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus [9] . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses [10, 11] . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus (RHDV), which is the pathogen causing Rabbit haemorrhagic disease (RHD), also known as rabbit calicivirus disease (RCD) or viral haemorrhagic disease (VHD), a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames (ORFs) separately encoding a predicted polyprotein and a minor structural protein named VP10 [12] . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 [13, 14] . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 [15] . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: (i) the extent and causes of codon bias in RHDV; (ii) A possible genotyping of RHDV; (iii) Codon usage bias as a factor reducing the expression of VP10 and (iiii) the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number (SN), collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports (2 Sharp, Tuohy et al. 1986 ) as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias [16] , was calculated for the quantification of the codon usage bias of each ORF [17] . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given (G+C) 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program [18] . Analyses were conducted with the Nei-Gojobori model [19] , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω (dn/ds) were calculated in MEGA4.0 [20] . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon (excluding Met, Trp, and the termination codons) [21] . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern [22] . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, (C+G)% content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and (C+G)% content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region (length > 250 bps) in the ORF1 of RHDV (30 isolates) genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω(dN/dS) values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis (f' 1 ) which accounted for 42.967% of the total variation, and another major trend in the second axis (f' 2 ) which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and (C+G)% were compared with A 3 %, U 3 %, C 3 %, G 3 % and (C 3 +G 3 )%, respectively (Table 5 ). There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes (f' 1 and f' 2 ) of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis (f' 1 ) accounts for 42.967% of the total variation, and the second axis (f' 2 ) accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV (Table 2) , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values [18] . ENC values of RHDV genomes were plotted against its corresponding (C 3 +G 3 ) %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 [5] . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome [23] [24] [25] [26] [27] [28] [29] , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 (Table 7) , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is positive value and the other one (marked as Group 2) is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference.

What factor may influence viral replication and gene expression?

Bioinformatics analysis of rabbit haemorrhagic disease virus genome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377956/ SHA: eff26d8739498efca2d32fe2e66cdbebf0569c50 Authors: Tian, Xiao-ting; Li, Bao-yu; Zhang, Liang; Jiao, Wen-qiang; Liu, Ji-xing Date: 2011-11-01 DOI: 10.1186/1743-422x-8-494 License: cc-by Abstract: BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly [1] . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors [2, 3] . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response [4] . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition [5] . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus [6] . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets [4] . Analysis of the bovine papillomavirus type 1 (BPV1) late genes has revealed a relationship between codon usage and tRNA availability [7] . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression [8] . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus [9] . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses [10, 11] . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus (RHDV), which is the pathogen causing Rabbit haemorrhagic disease (RHD), also known as rabbit calicivirus disease (RCD) or viral haemorrhagic disease (VHD), a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames (ORFs) separately encoding a predicted polyprotein and a minor structural protein named VP10 [12] . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 [13, 14] . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 [15] . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: (i) the extent and causes of codon bias in RHDV; (ii) A possible genotyping of RHDV; (iii) Codon usage bias as a factor reducing the expression of VP10 and (iiii) the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number (SN), collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports (2 Sharp, Tuohy et al. 1986 ) as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias [16] , was calculated for the quantification of the codon usage bias of each ORF [17] . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given (G+C) 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program [18] . Analyses were conducted with the Nei-Gojobori model [19] , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω (dn/ds) were calculated in MEGA4.0 [20] . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon (excluding Met, Trp, and the termination codons) [21] . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern [22] . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, (C+G)% content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and (C+G)% content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region (length > 250 bps) in the ORF1 of RHDV (30 isolates) genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω(dN/dS) values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis (f' 1 ) which accounted for 42.967% of the total variation, and another major trend in the second axis (f' 2 ) which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and (C+G)% were compared with A 3 %, U 3 %, C 3 %, G 3 % and (C 3 +G 3 )%, respectively (Table 5 ). There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes (f' 1 and f' 2 ) of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis (f' 1 ) accounts for 42.967% of the total variation, and the second axis (f' 2 ) accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV (Table 2) , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values [18] . ENC values of RHDV genomes were plotted against its corresponding (C 3 +G 3 ) %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 [5] . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome [23] [24] [25] [26] [27] [28] [29] , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 (Table 7) , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is positive value and the other one (marked as Group 2) is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference.

What accounts for the variation of codon usage among open reading frameworks?

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.

What conditions are considered upper respiratory tracts infections?

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.

What conditions are considered lower respiratory tract infections?

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.

What immune cells are primarily involved in eliminating virus-infected cells?

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.

What molecules have been shown to hinder T cell responses to viral infections?

Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.

The accumulation of what molecule hinders phagocytic activity in T cells?

A Global Champion for Health—WHO’s Next? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924837/ SHA: f2f9088055600d4160e36db5cb6ea000916390a3 Authors: nan Date: 2016-06-28 DOI: 10.1371/journal.pmed.1002059 License: cc-by Abstract: In this month’s editorial, the PLOS Medicine Editors propose ideal qualities for the World Health Organization's next Director General, for whom the selection process is now underway. Text: response to the Ebola outbreak [1] . Reformation of WHO to ready it to lead responses to future health emergencies is one area of active debate. Chan will step down from WHO on June 30, 2017 after more than a decade in the post. The process for choosing WHO's next leader has begun, promising to be protracted and rigorous as befits the importance of the role. Factoring in the many influential stakeholders in the process of appointing Chan's successor, however, transparency of the selection process may be one area unlikely to attract plaudits. Although too soon to speculate about the identity of WHO's next Director-General, it is worth reflecting on what qualities an incoming leader should bring to WHO and how that person might need to conceive changes in the structure and behavior of the organization against a landscape of important and evolving threats to the health of the fastgrowing global population. Instead of electing a new Director-General, Lorenz Von Seidlein of Mahidol University, Thailand, argued that "the problems. . .are now so deeply ingrained that replacing the WHO with new, more appropriate organizations is the logical solution. . .at a fraction of current cost, free of cumbersome, archaic obligations and entitlements and [with] an ability to respond to new problems." This viewpoint is indicative of the strength of feeling that WHO's deficiencies have come to evoke in some of those committed to the cause of improving the health of people in low-income and middle-income countries. But this perception acknowledges that an accountable global body will always be needed to promote, set standards in, and evaluate progress toward better health for people in all countries. The next Director-General will need to heed critics of the organization and craft a process of streamlining and restructuring to produce a new WHO that is demonstrably effective in leading responses to threats to health, and efficient in doing so. As Gostin commented to PLOS Medicine, "WHO urgently needs a bold reform agenda to fix long-standing problems recognized by every independent group that has evaluated the Organization." Political machinations and the enemy within, bureaucracy, are likely to impede reform. For example, WHO's regional and country offices are seen by some as unaccountable, yet the agency of the future will need to be connected and responsive to the resources and needs of all constituent countries. As Gostin also noted, "[WHO] has failed to include civil society in its governance, unlike. . .newer organizations." WHO's next Director-General should be a proven leader and advocate, perhaps from a lowincome or middle-income country. The new recruit will be greeted by a full in-tray, and featuring prominently are likely to be the constraints imposed by WHO's current funding mechanisms. A substantial proportion of WHO's existing budget is earmarked for specific projects, leaving the organization with little financial flexibility to respond to unanticipated demands. However, any improved funding mechanism is likely to follow, and be dependent on, organizational reform. According to Kruk, "WHO is both essential and hamstrung. . .the election of the Director-General should be a moment for member countries and other funders to reflect on whether they want an implementation agency for their favored health agenda, or an independent institution with the intelligence, agility, and operational capacity to tackle the coming global health challenges." Above all, the incoming leader of WHO will need to be open-minded and creative. More than one of the experts we contacted emphasized the fluid nature of the threats to human health to which WHO should shape the world's response. WHO must be able to lead responses in some areas of global health, but, in other areas, working together with more nimble and focused organizations will be pragmatic. Large-scale infectious disease outbreaks are continuing, and noncommunicable diseases, including cancer, dementia, and mental illnesses, are growing in prevalence and increasing demand for treatment and care. The resources and ingenuity of researchers and clinicians will need to be harnessed, and interventions adapted to new settings, with much greater dynamism. The secular issues of population ageing, conflict, climate change, migration, and others will produce health problems that only an organization with a global reach, responsible to all, can hope to meet. We look forward to welcoming a new leader for WHO with the energy and vision to remold the organization to meet the health needs of the world's people and societies for the 21st century.

When did the last Director General of the WHO resign?

A Global Champion for Health—WHO’s Next? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924837/ SHA: f2f9088055600d4160e36db5cb6ea000916390a3 Authors: nan Date: 2016-06-28 DOI: 10.1371/journal.pmed.1002059 License: cc-by Abstract: In this month’s editorial, the PLOS Medicine Editors propose ideal qualities for the World Health Organization's next Director General, for whom the selection process is now underway. Text: response to the Ebola outbreak [1] . Reformation of WHO to ready it to lead responses to future health emergencies is one area of active debate. Chan will step down from WHO on June 30, 2017 after more than a decade in the post. The process for choosing WHO's next leader has begun, promising to be protracted and rigorous as befits the importance of the role. Factoring in the many influential stakeholders in the process of appointing Chan's successor, however, transparency of the selection process may be one area unlikely to attract plaudits. Although too soon to speculate about the identity of WHO's next Director-General, it is worth reflecting on what qualities an incoming leader should bring to WHO and how that person might need to conceive changes in the structure and behavior of the organization against a landscape of important and evolving threats to the health of the fastgrowing global population. Instead of electing a new Director-General, Lorenz Von Seidlein of Mahidol University, Thailand, argued that "the problems. . .are now so deeply ingrained that replacing the WHO with new, more appropriate organizations is the logical solution. . .at a fraction of current cost, free of cumbersome, archaic obligations and entitlements and [with] an ability to respond to new problems." This viewpoint is indicative of the strength of feeling that WHO's deficiencies have come to evoke in some of those committed to the cause of improving the health of people in low-income and middle-income countries. But this perception acknowledges that an accountable global body will always be needed to promote, set standards in, and evaluate progress toward better health for people in all countries. The next Director-General will need to heed critics of the organization and craft a process of streamlining and restructuring to produce a new WHO that is demonstrably effective in leading responses to threats to health, and efficient in doing so. As Gostin commented to PLOS Medicine, "WHO urgently needs a bold reform agenda to fix long-standing problems recognized by every independent group that has evaluated the Organization." Political machinations and the enemy within, bureaucracy, are likely to impede reform. For example, WHO's regional and country offices are seen by some as unaccountable, yet the agency of the future will need to be connected and responsive to the resources and needs of all constituent countries. As Gostin also noted, "[WHO] has failed to include civil society in its governance, unlike. . .newer organizations." WHO's next Director-General should be a proven leader and advocate, perhaps from a lowincome or middle-income country. The new recruit will be greeted by a full in-tray, and featuring prominently are likely to be the constraints imposed by WHO's current funding mechanisms. A substantial proportion of WHO's existing budget is earmarked for specific projects, leaving the organization with little financial flexibility to respond to unanticipated demands. However, any improved funding mechanism is likely to follow, and be dependent on, organizational reform. According to Kruk, "WHO is both essential and hamstrung. . .the election of the Director-General should be a moment for member countries and other funders to reflect on whether they want an implementation agency for their favored health agenda, or an independent institution with the intelligence, agility, and operational capacity to tackle the coming global health challenges." Above all, the incoming leader of WHO will need to be open-minded and creative. More than one of the experts we contacted emphasized the fluid nature of the threats to human health to which WHO should shape the world's response. WHO must be able to lead responses in some areas of global health, but, in other areas, working together with more nimble and focused organizations will be pragmatic. Large-scale infectious disease outbreaks are continuing, and noncommunicable diseases, including cancer, dementia, and mental illnesses, are growing in prevalence and increasing demand for treatment and care. The resources and ingenuity of researchers and clinicians will need to be harnessed, and interventions adapted to new settings, with much greater dynamism. The secular issues of population ageing, conflict, climate change, migration, and others will produce health problems that only an organization with a global reach, responsible to all, can hope to meet. We look forward to welcoming a new leader for WHO with the energy and vision to remold the organization to meet the health needs of the world's people and societies for the 21st century.

Why might an organization like the WHO be necessary?

A Global Champion for Health—WHO’s Next? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924837/ SHA: f2f9088055600d4160e36db5cb6ea000916390a3 Authors: nan Date: 2016-06-28 DOI: 10.1371/journal.pmed.1002059 License: cc-by Abstract: In this month’s editorial, the PLOS Medicine Editors propose ideal qualities for the World Health Organization's next Director General, for whom the selection process is now underway. Text: response to the Ebola outbreak [1] . Reformation of WHO to ready it to lead responses to future health emergencies is one area of active debate. Chan will step down from WHO on June 30, 2017 after more than a decade in the post. The process for choosing WHO's next leader has begun, promising to be protracted and rigorous as befits the importance of the role. Factoring in the many influential stakeholders in the process of appointing Chan's successor, however, transparency of the selection process may be one area unlikely to attract plaudits. Although too soon to speculate about the identity of WHO's next Director-General, it is worth reflecting on what qualities an incoming leader should bring to WHO and how that person might need to conceive changes in the structure and behavior of the organization against a landscape of important and evolving threats to the health of the fastgrowing global population. Instead of electing a new Director-General, Lorenz Von Seidlein of Mahidol University, Thailand, argued that "the problems. . .are now so deeply ingrained that replacing the WHO with new, more appropriate organizations is the logical solution. . .at a fraction of current cost, free of cumbersome, archaic obligations and entitlements and [with] an ability to respond to new problems." This viewpoint is indicative of the strength of feeling that WHO's deficiencies have come to evoke in some of those committed to the cause of improving the health of people in low-income and middle-income countries. But this perception acknowledges that an accountable global body will always be needed to promote, set standards in, and evaluate progress toward better health for people in all countries. The next Director-General will need to heed critics of the organization and craft a process of streamlining and restructuring to produce a new WHO that is demonstrably effective in leading responses to threats to health, and efficient in doing so. As Gostin commented to PLOS Medicine, "WHO urgently needs a bold reform agenda to fix long-standing problems recognized by every independent group that has evaluated the Organization." Political machinations and the enemy within, bureaucracy, are likely to impede reform. For example, WHO's regional and country offices are seen by some as unaccountable, yet the agency of the future will need to be connected and responsive to the resources and needs of all constituent countries. As Gostin also noted, "[WHO] has failed to include civil society in its governance, unlike. . .newer organizations." WHO's next Director-General should be a proven leader and advocate, perhaps from a lowincome or middle-income country. The new recruit will be greeted by a full in-tray, and featuring prominently are likely to be the constraints imposed by WHO's current funding mechanisms. A substantial proportion of WHO's existing budget is earmarked for specific projects, leaving the organization with little financial flexibility to respond to unanticipated demands. However, any improved funding mechanism is likely to follow, and be dependent on, organizational reform. According to Kruk, "WHO is both essential and hamstrung. . .the election of the Director-General should be a moment for member countries and other funders to reflect on whether they want an implementation agency for their favored health agenda, or an independent institution with the intelligence, agility, and operational capacity to tackle the coming global health challenges." Above all, the incoming leader of WHO will need to be open-minded and creative. More than one of the experts we contacted emphasized the fluid nature of the threats to human health to which WHO should shape the world's response. WHO must be able to lead responses in some areas of global health, but, in other areas, working together with more nimble and focused organizations will be pragmatic. Large-scale infectious disease outbreaks are continuing, and noncommunicable diseases, including cancer, dementia, and mental illnesses, are growing in prevalence and increasing demand for treatment and care. The resources and ingenuity of researchers and clinicians will need to be harnessed, and interventions adapted to new settings, with much greater dynamism. The secular issues of population ageing, conflict, climate change, migration, and others will produce health problems that only an organization with a global reach, responsible to all, can hope to meet. We look forward to welcoming a new leader for WHO with the energy and vision to remold the organization to meet the health needs of the world's people and societies for the 21st century.

Where should the next Director General for the WHO come from?

A Global Champion for Health—WHO’s Next? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924837/ SHA: f2f9088055600d4160e36db5cb6ea000916390a3 Authors: nan Date: 2016-06-28 DOI: 10.1371/journal.pmed.1002059 License: cc-by Abstract: In this month’s editorial, the PLOS Medicine Editors propose ideal qualities for the World Health Organization's next Director General, for whom the selection process is now underway. Text: response to the Ebola outbreak [1] . Reformation of WHO to ready it to lead responses to future health emergencies is one area of active debate. Chan will step down from WHO on June 30, 2017 after more than a decade in the post. The process for choosing WHO's next leader has begun, promising to be protracted and rigorous as befits the importance of the role. Factoring in the many influential stakeholders in the process of appointing Chan's successor, however, transparency of the selection process may be one area unlikely to attract plaudits. Although too soon to speculate about the identity of WHO's next Director-General, it is worth reflecting on what qualities an incoming leader should bring to WHO and how that person might need to conceive changes in the structure and behavior of the organization against a landscape of important and evolving threats to the health of the fastgrowing global population. Instead of electing a new Director-General, Lorenz Von Seidlein of Mahidol University, Thailand, argued that "the problems. . .are now so deeply ingrained that replacing the WHO with new, more appropriate organizations is the logical solution. . .at a fraction of current cost, free of cumbersome, archaic obligations and entitlements and [with] an ability to respond to new problems." This viewpoint is indicative of the strength of feeling that WHO's deficiencies have come to evoke in some of those committed to the cause of improving the health of people in low-income and middle-income countries. But this perception acknowledges that an accountable global body will always be needed to promote, set standards in, and evaluate progress toward better health for people in all countries. The next Director-General will need to heed critics of the organization and craft a process of streamlining and restructuring to produce a new WHO that is demonstrably effective in leading responses to threats to health, and efficient in doing so. As Gostin commented to PLOS Medicine, "WHO urgently needs a bold reform agenda to fix long-standing problems recognized by every independent group that has evaluated the Organization." Political machinations and the enemy within, bureaucracy, are likely to impede reform. For example, WHO's regional and country offices are seen by some as unaccountable, yet the agency of the future will need to be connected and responsive to the resources and needs of all constituent countries. As Gostin also noted, "[WHO] has failed to include civil society in its governance, unlike. . .newer organizations." WHO's next Director-General should be a proven leader and advocate, perhaps from a lowincome or middle-income country. The new recruit will be greeted by a full in-tray, and featuring prominently are likely to be the constraints imposed by WHO's current funding mechanisms. A substantial proportion of WHO's existing budget is earmarked for specific projects, leaving the organization with little financial flexibility to respond to unanticipated demands. However, any improved funding mechanism is likely to follow, and be dependent on, organizational reform. According to Kruk, "WHO is both essential and hamstrung. . .the election of the Director-General should be a moment for member countries and other funders to reflect on whether they want an implementation agency for their favored health agenda, or an independent institution with the intelligence, agility, and operational capacity to tackle the coming global health challenges." Above all, the incoming leader of WHO will need to be open-minded and creative. More than one of the experts we contacted emphasized the fluid nature of the threats to human health to which WHO should shape the world's response. WHO must be able to lead responses in some areas of global health, but, in other areas, working together with more nimble and focused organizations will be pragmatic. Large-scale infectious disease outbreaks are continuing, and noncommunicable diseases, including cancer, dementia, and mental illnesses, are growing in prevalence and increasing demand for treatment and care. The resources and ingenuity of researchers and clinicians will need to be harnessed, and interventions adapted to new settings, with much greater dynamism. The secular issues of population ageing, conflict, climate change, migration, and others will produce health problems that only an organization with a global reach, responsible to all, can hope to meet. We look forward to welcoming a new leader for WHO with the energy and vision to remold the organization to meet the health needs of the world's people and societies for the 21st century.

What traits should the new Director General of the WHO have?

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852297/ SHA: ee8dca216514deeed4c9415bc2ad8a78dc3d9670 Authors: Fournier, Carole; Duverlie, Gilles; François, Catherine; Schnuriger, Aurelie; Dedeurwaerder, Sarah; Brochot, Etienne; Capron, Dominique; Wychowski, Czeslaw; Thibault, Vincent; Castelain, Sandrine Date: 2007-03-30 DOI: 10.1186/1743-422x-4-35 License: cc-by Abstract: BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies. Text: Hepatitis C virus (HCV, a member of the Flaviviridae family) is an enveloped, positive-stranded RNA virus that preferentially replicates in hepatocytes. At least 170 million people worldwide are persistently infected with hepatitis C virus. Chronic HCV infection is associated with a significant risk of progression to cirrhosis and hepatocellular carcinoma [1] . Antiviral therapy with pegylated alpha-interferon and ribavirin (the current best therapeutic regimen) is only successful in about 50% of all treated patients. Better knowledge of the viral and host factors that determine HCV clearance or persistence during the acute stage of infection is needed in order to improve antiviral therapy and to develop efficient vaccines. Studies focusing on innate and cellular immune responses have shown that a sufficiently large HCV inoculum is able to evade, subvert or circumvent the host's defences. At present, the chimpanzee is the only reliable experimental animal model in which the initial post-HCV infection events and the efficacy of vaccine candidates can be evaluated [2] . It has been shown that HCV-specific T-cell immunity is important in the control of HCV infection [3, 4] . Several studies have indicated a role for humoral immunity in the acute stage of HCV infection but this aspect remains poorly characterized. The E1 and E2 glycoproteins are thought to be the viral attachment proteins and thus the main targets for HCV-neutralizing antibodies; identification of protective epitopes conserved across different strains of HCV is therefore a major challenge in vaccine design. A number of antibodies capable of blocking E2 binding to cells or cell receptors have been described, [5] [6] [7] [8] some of which neutralize HCV entry in animal or cellular models [9, 10] . Cell entry has been shown to involve several surface molecules (notably including the tetraspanin CD81 and the SR-BI receptor [11, 12] ), although further studies are needed to better understand how viral entry occurs and how it might be neutralized. Detection of neutralizing antibodies in human blood had been problematical until an efficient and reliable cell culture system for HCV became available. Hence, the development of an in vitro neutralization assay for HCV could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential of passive and active immunization against hepatitis C. Recent studies using an in vitro neutralization assay system (based on infectious retroviral pseudoparticles (HCVpp) bearing HCV envelope glycoproteins) have confirmed that HCV-infected patient sera can indeed neutralize infection [13, 14] . However, it has also been shown that the neutralizing activity of antibodies from HCV-infected patients is attenuated by a factor present in human serum, identified as the highdensity lipoprotein (HDL) fraction [11, 13, 15] . HDL facilitation of HCVpp entry is a post-binding event [16] , sug-gesting that HDLs favour internalization of virions and thus the latter's escape from neutralizing antibodies. Recently, an HCV cell culture model (HCVcc) has been developed [17] [18] [19] , allowing the production of virus particles that can be efficiently propagated in cell culture. Some preliminary neutralization assays have been carried out by these authors. In this study, we describe how we set up a standardized focus reduction neutralization assay based on HCVcc. Focus reduction assays have been widely used to evaluate the neutralizing antibody responses to viruses that can form foci in infected cells. Following the recent development of the HCVcc model, the principle of the focus reduction assay has been applied to HCV-neutralizing antibodies detection. The JFH-1 HCV 2a viral strain was grown on a Huh-7 human hepatoma cell line. After three days of infection and cell permeabilization, detection of the HCV foci was carried out using an inactivated HCVpositive patient serum primary antibody and a peroxidase-coupled, Fc-specific anti-human IgG-antibody. The reaction was revealed with DAB peroxidase substrate. The viral foci were thus stained brown, making them easy to count (see Fig. 1a ). It has been recently shown that the neutralizing activity of HCV antibodies is attenuated by a serum factor associated with the HDL fraction. Hence, HDLs were able to facilitate HCVpp and HCVcc entry via a mechanism which depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function [11, 15, 16, 20] . In view of the role of HDL in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibody titer (see Fig. 1b ). The specificity of the HCV neutralization assay was assessed by testing 20 anti-HCV-ELISA-negative samples, including five positive for hepatitis B virus surface antibodies (anti-HBs) and five positive for heterophile antibodies. All samples tested negative with two commercial anti-HCV antibody detection assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany; Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom) and HCV-RNA-negative with a qualitative, commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France). These anti-HCV-negative samples were compared with 11 samples from patients chronically infected with HCV genotype 2. The neutralization titers of anti-HCV-negative serum samples are shown in Fig. 2 ., with a mean value of 1.083 ± 0.083 (corresponding to a dilution of 1:12). The assay's cut-off (determined as the mean value for negative samples plus two standard deviations) corresponded to a dilution of 1:18. The assay exhibited specificity and sensibility values of 100% and 96.5%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples (data not shown). Conversely, the chronically HCV genotype 2-positive samples displayed strong reactions, with a mean value of 2.128 ± 0.365 (corresponding to a dilution of 1:134) (p < 0.001). Inter-assay variability was determined by testing one HCV genotype 2 sample in 10 consecutive experiments (n = 10), whereas intra-assay variability was evaluated by testing the same sample 10 times (n = 10) in the same experiment, whilst running the dilution series. The intra-assay and inter-assay coefficients of variation (CV) of the log neutralization titers were 6.7% and 12.6%, respectively. Fifty-seven HCV-positive antibodies samples were evaluated using the HCV focus reduction neutralization assay. The genotypes were distributed as follows; for types 1a, 1b, 2, 3, 4 and 5, we studied 11, 11, 11, 12, 10 and 2 samples, respectively. The mean values of the different genotypes is shown in Fig. 3 . and Table 1 . The mean log neutralization titers for genotypes 1a, 2 and 3 are very similar (2.046 ± 0.671 for genotype 1a, 2.128 ± 0.365 for genotype 2 and 2.148 ± 0.478 for genotype 3). The mean average values are lower for genotype 1b (1.747 ± 0.462) and genotype 4 (1.786 ± 0.236). Strikingly, very high heterologous titers were observed for five patients -three infected with HCV genotype 1a and two infected with HCV genotype 3 (see Fig. 3a ). There were too few genotype 5 samples to compare with the other genotypes but the corresponding results nevertheless indicate that the neutralization assay is suitable for this genotype. The two The distribution of the log neutralization titers across all the HCV ELISA and RNA-positive samples as a function of the HCV genotype is shown in Fig. 3b . More than 60% of the neutralizing antibodies titers fell in the range from 1.7 to 2.69 log titers, corresponding to dilutions of 1:50 and 1:500, respectively. Overall, 3.5% of the samples displayed a titer greater than log 3.0 (1:1000) and, conversely, 3.5% displayed a titer below the cut-off value, i.e. log 1.25 (1:10). Thus, of 57 HCV-infected patients, only two did not test positive for neutralizing antibodies in this assay (the titers were 0.960 and 0.932, respectively). The role of neutralizing antibodies during acute and chronic viral infection remains an important question and has generated controversial results. Initially, the presence of neutralizing antibodies was shown to control the HCV load and to contribute to viral eradication in patients capable of clearing the infection [13] . In other studies, the appearance of neutralizing antibodies was delayed and restricted to IgG1 antibodies in patients who develop a chronic infection [2, 21] . The chimpanzee model has been critical for the study of HCV transmission and host immune responses; however, neutralizing antibodies were not detected in some animals that resolved their infection -suggesting a minimal role in viral clearance, as also observed in human studies [14, 15] . Experimentally infected chimpanzees and naturally infected humans can be re-infected with homologous and heterologous HCV strains, suggesting that the humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing [22] [23] [24] . During chronic infection in humans, the presence and/or production of neutralizing antibodies do not suffice for curing the infection but could regulate the spread of the virus. Thus, it can be postulated that during chronic infection, viral mutants can continuously escape the renewed production of neutralizing antibodies. Retroviral pseudoparticles have been used to develop a very interesting tool for measuring neutralizing antibodies in vitro [14] . The assay has demonstrated the presence of HCV-neutralizing antibodies in human sera with relatively high titers (>1:320) and broadly neutralizing activity against different HCV genotypes. However, this model does not represent genuine HCV virions; in particular, the budding of retroviral particles is thought to be very different and may involve a variety of cellular pathways. Characterization of infectious retroviral pseudotype particles bearing HCV glycoproteins have been shown to be very heterogeneous, and so it is possible that these pseudoparticles may not be as relevant as the native HCV virions [25] . The recent development of a cell culture model for HCV enables the production of native HCV virions that can be efficiently propagated in cell culture [17] [18] [19] . This cell culture system has allowed us to develop a neutralization assay for evaluating the level and the proportion of HCVneutralizing antibodies in chronically infected HCV patients. We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26] [27] [28] . The assay relies on the ability of the specific JFH-1 genotype 2 viral strain to replicate and multiply on a Huh-7 human hepatoma cell line in a cell culture model, enabling the rapid detection of viral foci after 72 hours of infection. Moreover, no secondary foci were detectable at this time point. Fixation with paraformaldehyde and permeabilization with Triton X-100 were chosen in order to preserve antigenicity and prevent the cell monolayer from detaching during washes. Development with DAB peroxide substrate made it easy to count specifically coloured viral foci. The viral inoculum size is an important parameter; it has to be low enough to enable good assay sensitivity but high enough to produce a statistically significant number of foci, i.e. allowing the reduction in the number of foci (and thus the effect of neutralization) to be monitored. Thus, 100 FFUs were used as the inoculum in this neutralization assay. In order to test different human samples, we had to take into account the ability of HDL to facilitate HCVcc entry via a mechanism which depends on expression of the scavenger receptor BI [11, 15, 16, 20] . Given HDL's role in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibodies titer; this frees the assay of the risk of non-specific neutralization activity of the serum via the effects of HDL, the complement system and/or serum amyloid A protein (SAA) [29] . The HCV neutralization assay exhibited good reproducibility, for both duplicate assays and independent tests. As expected, the intra-assay coefficient of variation (CV) was lower than the interassay CV. The test also showed good specificity, since there was no interaction with anti-HIV, anti-HBV or heterophile antibodies. Very low titers were found with HCV ELISA and RNA-negative samples, and the assay's cut-off was determined as the mean titer for negative samples plus two standard deviations (1.25 log, corresponding to a dilution of 1:18). Given that only the JFH-1 strain of HCV genotype 2a was available for the assay, we evaluated the neutralization titer of sera from patients chronically infected with other HCV genotypes, i.e. 1, 2, 3, 4 and 5. Most of these sera were detected as positive by the neutralization assay, except for two sera from HCV genotype 1-infected patients. These two samples presented a high specific antibody ratio according to the ELISA but only very low inhibition by neutralization assay (far below the cut-off, in fact). We conclude that either the samples lacked neutralizing antibodies or that any such antibodies that were present did not cross-neutralize with HCV genotype 2a. The sensitivity was 100% -not only for genotype 2 (the genotype of the strain used for the assay) but also for other HCV genotypes (except genotype 1). HCV genotype 5 antibodies were also measured but there were too few samples for accurate testing. Moreover, the positive sera (96.5%) had comparable and significantly high titers (1.99 ± 0.63), whatever the genotype. This finding suggests that most neutralizing antibodies are cross-reactive. Another possibility is that most of the patients had been previously infected by a genotype 2 strain. However, this is unlikely because few genotype 2 strains are circulating in France [30] . As expected for a neutralization test, the assay presented in the present study appeared to be very specific (independently of the genotype) and usable in most circumstances. For most viral infections, neutralization assays such as that described in this study are used as reference assays. Thus, we are confident that as other HCVcc genotypes become available, these assays will replace the pseudoparticle assay in the near future because they are probably more relevant. Our assay is somewhat time-consuming and could be simplified by using one dilution to count the foci; however, this type of "short cut" would make it difficult to extrapolate to the dilution neutralizing 50% of the inoculum. Another approach would consist in using recombinant HCV capable of expressing reporter genes (such as luciferase) in order to use a single dilution and obtain a quantitative result [31] . However, further neutralization studies using other genotypes are needed in order to complete our observations and to char- A simple, specific and reproducible cell culture-based neutralization assay was developed for the determination of neutralizing anti-HCV antibodies in human sera. This test should be an important tool for gauging the relationship between the neutralizing response and viral load kinetics in acutely and chronically infected patients. The Huh-7 human hepatoma cells [32] were grown in Dulbecco's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum. All cell cultures were maintained in 5% CO 2 at 37°C. The plasmid pJFH-1 containing the full-length cDNA of the JFH-1 isolate (which belongs to subtype 2a (GenBank accession no. AB047639)), was a gift from Dr Wakita (Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) and has been described previously [17] . To generate genomic HCV RNA, the plasmid pJFH-1 was linearized at the 3' end of the HCV cDNA and used as a template for in vitro transcription, as described previously [33] . Viral stocks were obtained by harvesting cell culture supernatants and freezing them at -80°C. Virus titration was performed on Huh-7 cells with 6-well microtiter plates (Corning, NY) 72 hours after incubation, by immunostaining the cells with antibodies from a HCV-positive patient serum that had previously been inactivated at 56°C (see the section on the virus neutralization assay). The viral titer was determined in triplicate from the mean number of foci and expressed as focus forming units/mL (FFU/mL). Seventy-seven human serum samples were tested. Collection of the sera was approved by the local Ethics Committee and informed consent had been obtained from the donors. Fifty-seven of these samples were obtained from chronically infected HCV patients. The presence of HCV antibodies was determined and confirmed using two third-generation HCV EIA assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany and Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom). HCV RNA was determined with a qualitative commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France) and HCV genotyping was performed by direct sequencing, as described elsewhere [34] . The genotypes were distributed as follows: 11, 11, 11, 12, 10 and 2 samples of types 1a, 1b, 2, 3, 4 and 5, respectively. A set of 20 anti-HCV-negative serum samples was used to evaluate the assay's specif-icity, including five serum samples with positive hepatitis B virus surface antibody (anti-HBs) status and five sera from Epstein-Barr virus-infected patients that had tested positive for heterophile antibodies. All serum samples had been stored at -80°C upon collection and had not been thawed until the time of assay. Serum immunoglobulins G (IgG) fraction was purified using protein G-Sepharose (GE Healthcare, Orsay, France The HCV focus reduction neutralization assay was performed in 96-well microtiter plates. Serial dilutions of purified IgG (10 μg) ranging from 1:10 to 1:1,280 were established. Each dilution was tested twice. 25 μL of each sample was mixed with 25 μL of virus (100 FFU) in 96well microtiter plates and incubated for 1 hour at 37°C, 5% CO 2 . A volume of 100 μL of Huh-7 cell suspension (10,000 cells/well) in culture medium was added and incubated for 5 hours at 37°C, 5% CO2. After 5 hours, the supernatants were removed and 100 μL of culture medium were added to the monolayers. After 72 hours, the cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Primary antibody (a HCVpositive patient serum inactivated at 56°C) was diluted to 1:500 prior to use and then incubated for 1 h at room temperature. A peroxidase-coupled, Fc-specific anti-human IgG antibody (Sigma, Saint Quentin Fallavier, France) diluted to 1:200 was dispensed onto the cell monolayer and incubated for 30 min at room temperature. The reaction was developed with DAB peroxidase substrate (Sigma, Saint Quentin Fallavier, France) and stopped after 10 min of incubation with distilled water. The number of HCV foci in each dilution was determined. Controls were included in each assay (non-neutralized virus, purified IgG from each patient at a 1:10 dilution). The dilution that neutralized 50% of the virus was calculated by curvilinear regression analysis using XLSTAT 2006 software (Addinsoft SARL, Paris, France) [35] . Each titer was deter-mined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. Titers were expressed as logarithmic values and means ± standard deviation were calculated. Student's t-test was used to compare data between groups. p values below 0.05 were considered to be significant.

What is the Hepatitis C virus?

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852297/ SHA: ee8dca216514deeed4c9415bc2ad8a78dc3d9670 Authors: Fournier, Carole; Duverlie, Gilles; François, Catherine; Schnuriger, Aurelie; Dedeurwaerder, Sarah; Brochot, Etienne; Capron, Dominique; Wychowski, Czeslaw; Thibault, Vincent; Castelain, Sandrine Date: 2007-03-30 DOI: 10.1186/1743-422x-4-35 License: cc-by Abstract: BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies. Text: Hepatitis C virus (HCV, a member of the Flaviviridae family) is an enveloped, positive-stranded RNA virus that preferentially replicates in hepatocytes. At least 170 million people worldwide are persistently infected with hepatitis C virus. Chronic HCV infection is associated with a significant risk of progression to cirrhosis and hepatocellular carcinoma [1] . Antiviral therapy with pegylated alpha-interferon and ribavirin (the current best therapeutic regimen) is only successful in about 50% of all treated patients. Better knowledge of the viral and host factors that determine HCV clearance or persistence during the acute stage of infection is needed in order to improve antiviral therapy and to develop efficient vaccines. Studies focusing on innate and cellular immune responses have shown that a sufficiently large HCV inoculum is able to evade, subvert or circumvent the host's defences. At present, the chimpanzee is the only reliable experimental animal model in which the initial post-HCV infection events and the efficacy of vaccine candidates can be evaluated [2] . It has been shown that HCV-specific T-cell immunity is important in the control of HCV infection [3, 4] . Several studies have indicated a role for humoral immunity in the acute stage of HCV infection but this aspect remains poorly characterized. The E1 and E2 glycoproteins are thought to be the viral attachment proteins and thus the main targets for HCV-neutralizing antibodies; identification of protective epitopes conserved across different strains of HCV is therefore a major challenge in vaccine design. A number of antibodies capable of blocking E2 binding to cells or cell receptors have been described, [5] [6] [7] [8] some of which neutralize HCV entry in animal or cellular models [9, 10] . Cell entry has been shown to involve several surface molecules (notably including the tetraspanin CD81 and the SR-BI receptor [11, 12] ), although further studies are needed to better understand how viral entry occurs and how it might be neutralized. Detection of neutralizing antibodies in human blood had been problematical until an efficient and reliable cell culture system for HCV became available. Hence, the development of an in vitro neutralization assay for HCV could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential of passive and active immunization against hepatitis C. Recent studies using an in vitro neutralization assay system (based on infectious retroviral pseudoparticles (HCVpp) bearing HCV envelope glycoproteins) have confirmed that HCV-infected patient sera can indeed neutralize infection [13, 14] . However, it has also been shown that the neutralizing activity of antibodies from HCV-infected patients is attenuated by a factor present in human serum, identified as the highdensity lipoprotein (HDL) fraction [11, 13, 15] . HDL facilitation of HCVpp entry is a post-binding event [16] , sug-gesting that HDLs favour internalization of virions and thus the latter's escape from neutralizing antibodies. Recently, an HCV cell culture model (HCVcc) has been developed [17] [18] [19] , allowing the production of virus particles that can be efficiently propagated in cell culture. Some preliminary neutralization assays have been carried out by these authors. In this study, we describe how we set up a standardized focus reduction neutralization assay based on HCVcc. Focus reduction assays have been widely used to evaluate the neutralizing antibody responses to viruses that can form foci in infected cells. Following the recent development of the HCVcc model, the principle of the focus reduction assay has been applied to HCV-neutralizing antibodies detection. The JFH-1 HCV 2a viral strain was grown on a Huh-7 human hepatoma cell line. After three days of infection and cell permeabilization, detection of the HCV foci was carried out using an inactivated HCVpositive patient serum primary antibody and a peroxidase-coupled, Fc-specific anti-human IgG-antibody. The reaction was revealed with DAB peroxidase substrate. The viral foci were thus stained brown, making them easy to count (see Fig. 1a ). It has been recently shown that the neutralizing activity of HCV antibodies is attenuated by a serum factor associated with the HDL fraction. Hence, HDLs were able to facilitate HCVpp and HCVcc entry via a mechanism which depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function [11, 15, 16, 20] . In view of the role of HDL in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibody titer (see Fig. 1b ). The specificity of the HCV neutralization assay was assessed by testing 20 anti-HCV-ELISA-negative samples, including five positive for hepatitis B virus surface antibodies (anti-HBs) and five positive for heterophile antibodies. All samples tested negative with two commercial anti-HCV antibody detection assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany; Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom) and HCV-RNA-negative with a qualitative, commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France). These anti-HCV-negative samples were compared with 11 samples from patients chronically infected with HCV genotype 2. The neutralization titers of anti-HCV-negative serum samples are shown in Fig. 2 ., with a mean value of 1.083 ± 0.083 (corresponding to a dilution of 1:12). The assay's cut-off (determined as the mean value for negative samples plus two standard deviations) corresponded to a dilution of 1:18. The assay exhibited specificity and sensibility values of 100% and 96.5%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples (data not shown). Conversely, the chronically HCV genotype 2-positive samples displayed strong reactions, with a mean value of 2.128 ± 0.365 (corresponding to a dilution of 1:134) (p < 0.001). Inter-assay variability was determined by testing one HCV genotype 2 sample in 10 consecutive experiments (n = 10), whereas intra-assay variability was evaluated by testing the same sample 10 times (n = 10) in the same experiment, whilst running the dilution series. The intra-assay and inter-assay coefficients of variation (CV) of the log neutralization titers were 6.7% and 12.6%, respectively. Fifty-seven HCV-positive antibodies samples were evaluated using the HCV focus reduction neutralization assay. The genotypes were distributed as follows; for types 1a, 1b, 2, 3, 4 and 5, we studied 11, 11, 11, 12, 10 and 2 samples, respectively. The mean values of the different genotypes is shown in Fig. 3 . and Table 1 . The mean log neutralization titers for genotypes 1a, 2 and 3 are very similar (2.046 ± 0.671 for genotype 1a, 2.128 ± 0.365 for genotype 2 and 2.148 ± 0.478 for genotype 3). The mean average values are lower for genotype 1b (1.747 ± 0.462) and genotype 4 (1.786 ± 0.236). Strikingly, very high heterologous titers were observed for five patients -three infected with HCV genotype 1a and two infected with HCV genotype 3 (see Fig. 3a ). There were too few genotype 5 samples to compare with the other genotypes but the corresponding results nevertheless indicate that the neutralization assay is suitable for this genotype. The two The distribution of the log neutralization titers across all the HCV ELISA and RNA-positive samples as a function of the HCV genotype is shown in Fig. 3b . More than 60% of the neutralizing antibodies titers fell in the range from 1.7 to 2.69 log titers, corresponding to dilutions of 1:50 and 1:500, respectively. Overall, 3.5% of the samples displayed a titer greater than log 3.0 (1:1000) and, conversely, 3.5% displayed a titer below the cut-off value, i.e. log 1.25 (1:10). Thus, of 57 HCV-infected patients, only two did not test positive for neutralizing antibodies in this assay (the titers were 0.960 and 0.932, respectively). The role of neutralizing antibodies during acute and chronic viral infection remains an important question and has generated controversial results. Initially, the presence of neutralizing antibodies was shown to control the HCV load and to contribute to viral eradication in patients capable of clearing the infection [13] . In other studies, the appearance of neutralizing antibodies was delayed and restricted to IgG1 antibodies in patients who develop a chronic infection [2, 21] . The chimpanzee model has been critical for the study of HCV transmission and host immune responses; however, neutralizing antibodies were not detected in some animals that resolved their infection -suggesting a minimal role in viral clearance, as also observed in human studies [14, 15] . Experimentally infected chimpanzees and naturally infected humans can be re-infected with homologous and heterologous HCV strains, suggesting that the humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing [22] [23] [24] . During chronic infection in humans, the presence and/or production of neutralizing antibodies do not suffice for curing the infection but could regulate the spread of the virus. Thus, it can be postulated that during chronic infection, viral mutants can continuously escape the renewed production of neutralizing antibodies. Retroviral pseudoparticles have been used to develop a very interesting tool for measuring neutralizing antibodies in vitro [14] . The assay has demonstrated the presence of HCV-neutralizing antibodies in human sera with relatively high titers (>1:320) and broadly neutralizing activity against different HCV genotypes. However, this model does not represent genuine HCV virions; in particular, the budding of retroviral particles is thought to be very different and may involve a variety of cellular pathways. Characterization of infectious retroviral pseudotype particles bearing HCV glycoproteins have been shown to be very heterogeneous, and so it is possible that these pseudoparticles may not be as relevant as the native HCV virions [25] . The recent development of a cell culture model for HCV enables the production of native HCV virions that can be efficiently propagated in cell culture [17] [18] [19] . This cell culture system has allowed us to develop a neutralization assay for evaluating the level and the proportion of HCVneutralizing antibodies in chronically infected HCV patients. We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26] [27] [28] . The assay relies on the ability of the specific JFH-1 genotype 2 viral strain to replicate and multiply on a Huh-7 human hepatoma cell line in a cell culture model, enabling the rapid detection of viral foci after 72 hours of infection. Moreover, no secondary foci were detectable at this time point. Fixation with paraformaldehyde and permeabilization with Triton X-100 were chosen in order to preserve antigenicity and prevent the cell monolayer from detaching during washes. Development with DAB peroxide substrate made it easy to count specifically coloured viral foci. The viral inoculum size is an important parameter; it has to be low enough to enable good assay sensitivity but high enough to produce a statistically significant number of foci, i.e. allowing the reduction in the number of foci (and thus the effect of neutralization) to be monitored. Thus, 100 FFUs were used as the inoculum in this neutralization assay. In order to test different human samples, we had to take into account the ability of HDL to facilitate HCVcc entry via a mechanism which depends on expression of the scavenger receptor BI [11, 15, 16, 20] . Given HDL's role in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibodies titer; this frees the assay of the risk of non-specific neutralization activity of the serum via the effects of HDL, the complement system and/or serum amyloid A protein (SAA) [29] . The HCV neutralization assay exhibited good reproducibility, for both duplicate assays and independent tests. As expected, the intra-assay coefficient of variation (CV) was lower than the interassay CV. The test also showed good specificity, since there was no interaction with anti-HIV, anti-HBV or heterophile antibodies. Very low titers were found with HCV ELISA and RNA-negative samples, and the assay's cut-off was determined as the mean titer for negative samples plus two standard deviations (1.25 log, corresponding to a dilution of 1:18). Given that only the JFH-1 strain of HCV genotype 2a was available for the assay, we evaluated the neutralization titer of sera from patients chronically infected with other HCV genotypes, i.e. 1, 2, 3, 4 and 5. Most of these sera were detected as positive by the neutralization assay, except for two sera from HCV genotype 1-infected patients. These two samples presented a high specific antibody ratio according to the ELISA but only very low inhibition by neutralization assay (far below the cut-off, in fact). We conclude that either the samples lacked neutralizing antibodies or that any such antibodies that were present did not cross-neutralize with HCV genotype 2a. The sensitivity was 100% -not only for genotype 2 (the genotype of the strain used for the assay) but also for other HCV genotypes (except genotype 1). HCV genotype 5 antibodies were also measured but there were too few samples for accurate testing. Moreover, the positive sera (96.5%) had comparable and significantly high titers (1.99 ± 0.63), whatever the genotype. This finding suggests that most neutralizing antibodies are cross-reactive. Another possibility is that most of the patients had been previously infected by a genotype 2 strain. However, this is unlikely because few genotype 2 strains are circulating in France [30] . As expected for a neutralization test, the assay presented in the present study appeared to be very specific (independently of the genotype) and usable in most circumstances. For most viral infections, neutralization assays such as that described in this study are used as reference assays. Thus, we are confident that as other HCVcc genotypes become available, these assays will replace the pseudoparticle assay in the near future because they are probably more relevant. Our assay is somewhat time-consuming and could be simplified by using one dilution to count the foci; however, this type of "short cut" would make it difficult to extrapolate to the dilution neutralizing 50% of the inoculum. Another approach would consist in using recombinant HCV capable of expressing reporter genes (such as luciferase) in order to use a single dilution and obtain a quantitative result [31] . However, further neutralization studies using other genotypes are needed in order to complete our observations and to char- A simple, specific and reproducible cell culture-based neutralization assay was developed for the determination of neutralizing anti-HCV antibodies in human sera. This test should be an important tool for gauging the relationship between the neutralizing response and viral load kinetics in acutely and chronically infected patients. The Huh-7 human hepatoma cells [32] were grown in Dulbecco's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum. All cell cultures were maintained in 5% CO 2 at 37°C. The plasmid pJFH-1 containing the full-length cDNA of the JFH-1 isolate (which belongs to subtype 2a (GenBank accession no. AB047639)), was a gift from Dr Wakita (Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) and has been described previously [17] . To generate genomic HCV RNA, the plasmid pJFH-1 was linearized at the 3' end of the HCV cDNA and used as a template for in vitro transcription, as described previously [33] . Viral stocks were obtained by harvesting cell culture supernatants and freezing them at -80°C. Virus titration was performed on Huh-7 cells with 6-well microtiter plates (Corning, NY) 72 hours after incubation, by immunostaining the cells with antibodies from a HCV-positive patient serum that had previously been inactivated at 56°C (see the section on the virus neutralization assay). The viral titer was determined in triplicate from the mean number of foci and expressed as focus forming units/mL (FFU/mL). Seventy-seven human serum samples were tested. Collection of the sera was approved by the local Ethics Committee and informed consent had been obtained from the donors. Fifty-seven of these samples were obtained from chronically infected HCV patients. The presence of HCV antibodies was determined and confirmed using two third-generation HCV EIA assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany and Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom). HCV RNA was determined with a qualitative commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France) and HCV genotyping was performed by direct sequencing, as described elsewhere [34] . The genotypes were distributed as follows: 11, 11, 11, 12, 10 and 2 samples of types 1a, 1b, 2, 3, 4 and 5, respectively. A set of 20 anti-HCV-negative serum samples was used to evaluate the assay's specif-icity, including five serum samples with positive hepatitis B virus surface antibody (anti-HBs) status and five sera from Epstein-Barr virus-infected patients that had tested positive for heterophile antibodies. All serum samples had been stored at -80°C upon collection and had not been thawed until the time of assay. Serum immunoglobulins G (IgG) fraction was purified using protein G-Sepharose (GE Healthcare, Orsay, France The HCV focus reduction neutralization assay was performed in 96-well microtiter plates. Serial dilutions of purified IgG (10 μg) ranging from 1:10 to 1:1,280 were established. Each dilution was tested twice. 25 μL of each sample was mixed with 25 μL of virus (100 FFU) in 96well microtiter plates and incubated for 1 hour at 37°C, 5% CO 2 . A volume of 100 μL of Huh-7 cell suspension (10,000 cells/well) in culture medium was added and incubated for 5 hours at 37°C, 5% CO2. After 5 hours, the supernatants were removed and 100 μL of culture medium were added to the monolayers. After 72 hours, the cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Primary antibody (a HCVpositive patient serum inactivated at 56°C) was diluted to 1:500 prior to use and then incubated for 1 h at room temperature. A peroxidase-coupled, Fc-specific anti-human IgG antibody (Sigma, Saint Quentin Fallavier, France) diluted to 1:200 was dispensed onto the cell monolayer and incubated for 30 min at room temperature. The reaction was developed with DAB peroxidase substrate (Sigma, Saint Quentin Fallavier, France) and stopped after 10 min of incubation with distilled water. The number of HCV foci in each dilution was determined. Controls were included in each assay (non-neutralized virus, purified IgG from each patient at a 1:10 dilution). The dilution that neutralized 50% of the virus was calculated by curvilinear regression analysis using XLSTAT 2006 software (Addinsoft SARL, Paris, France) [35] . Each titer was deter-mined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. Titers were expressed as logarithmic values and means ± standard deviation were calculated. Student's t-test was used to compare data between groups. p values below 0.05 were considered to be significant.

How many people have persistent hepatitis C virus?

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852297/ SHA: ee8dca216514deeed4c9415bc2ad8a78dc3d9670 Authors: Fournier, Carole; Duverlie, Gilles; François, Catherine; Schnuriger, Aurelie; Dedeurwaerder, Sarah; Brochot, Etienne; Capron, Dominique; Wychowski, Czeslaw; Thibault, Vincent; Castelain, Sandrine Date: 2007-03-30 DOI: 10.1186/1743-422x-4-35 License: cc-by Abstract: BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies. Text: Hepatitis C virus (HCV, a member of the Flaviviridae family) is an enveloped, positive-stranded RNA virus that preferentially replicates in hepatocytes. At least 170 million people worldwide are persistently infected with hepatitis C virus. Chronic HCV infection is associated with a significant risk of progression to cirrhosis and hepatocellular carcinoma [1] . Antiviral therapy with pegylated alpha-interferon and ribavirin (the current best therapeutic regimen) is only successful in about 50% of all treated patients. Better knowledge of the viral and host factors that determine HCV clearance or persistence during the acute stage of infection is needed in order to improve antiviral therapy and to develop efficient vaccines. Studies focusing on innate and cellular immune responses have shown that a sufficiently large HCV inoculum is able to evade, subvert or circumvent the host's defences. At present, the chimpanzee is the only reliable experimental animal model in which the initial post-HCV infection events and the efficacy of vaccine candidates can be evaluated [2] . It has been shown that HCV-specific T-cell immunity is important in the control of HCV infection [3, 4] . Several studies have indicated a role for humoral immunity in the acute stage of HCV infection but this aspect remains poorly characterized. The E1 and E2 glycoproteins are thought to be the viral attachment proteins and thus the main targets for HCV-neutralizing antibodies; identification of protective epitopes conserved across different strains of HCV is therefore a major challenge in vaccine design. A number of antibodies capable of blocking E2 binding to cells or cell receptors have been described, [5] [6] [7] [8] some of which neutralize HCV entry in animal or cellular models [9, 10] . Cell entry has been shown to involve several surface molecules (notably including the tetraspanin CD81 and the SR-BI receptor [11, 12] ), although further studies are needed to better understand how viral entry occurs and how it might be neutralized. Detection of neutralizing antibodies in human blood had been problematical until an efficient and reliable cell culture system for HCV became available. Hence, the development of an in vitro neutralization assay for HCV could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential of passive and active immunization against hepatitis C. Recent studies using an in vitro neutralization assay system (based on infectious retroviral pseudoparticles (HCVpp) bearing HCV envelope glycoproteins) have confirmed that HCV-infected patient sera can indeed neutralize infection [13, 14] . However, it has also been shown that the neutralizing activity of antibodies from HCV-infected patients is attenuated by a factor present in human serum, identified as the highdensity lipoprotein (HDL) fraction [11, 13, 15] . HDL facilitation of HCVpp entry is a post-binding event [16] , sug-gesting that HDLs favour internalization of virions and thus the latter's escape from neutralizing antibodies. Recently, an HCV cell culture model (HCVcc) has been developed [17] [18] [19] , allowing the production of virus particles that can be efficiently propagated in cell culture. Some preliminary neutralization assays have been carried out by these authors. In this study, we describe how we set up a standardized focus reduction neutralization assay based on HCVcc. Focus reduction assays have been widely used to evaluate the neutralizing antibody responses to viruses that can form foci in infected cells. Following the recent development of the HCVcc model, the principle of the focus reduction assay has been applied to HCV-neutralizing antibodies detection. The JFH-1 HCV 2a viral strain was grown on a Huh-7 human hepatoma cell line. After three days of infection and cell permeabilization, detection of the HCV foci was carried out using an inactivated HCVpositive patient serum primary antibody and a peroxidase-coupled, Fc-specific anti-human IgG-antibody. The reaction was revealed with DAB peroxidase substrate. The viral foci were thus stained brown, making them easy to count (see Fig. 1a ). It has been recently shown that the neutralizing activity of HCV antibodies is attenuated by a serum factor associated with the HDL fraction. Hence, HDLs were able to facilitate HCVpp and HCVcc entry via a mechanism which depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function [11, 15, 16, 20] . In view of the role of HDL in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibody titer (see Fig. 1b ). The specificity of the HCV neutralization assay was assessed by testing 20 anti-HCV-ELISA-negative samples, including five positive for hepatitis B virus surface antibodies (anti-HBs) and five positive for heterophile antibodies. All samples tested negative with two commercial anti-HCV antibody detection assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany; Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom) and HCV-RNA-negative with a qualitative, commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France). These anti-HCV-negative samples were compared with 11 samples from patients chronically infected with HCV genotype 2. The neutralization titers of anti-HCV-negative serum samples are shown in Fig. 2 ., with a mean value of 1.083 ± 0.083 (corresponding to a dilution of 1:12). The assay's cut-off (determined as the mean value for negative samples plus two standard deviations) corresponded to a dilution of 1:18. The assay exhibited specificity and sensibility values of 100% and 96.5%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples (data not shown). Conversely, the chronically HCV genotype 2-positive samples displayed strong reactions, with a mean value of 2.128 ± 0.365 (corresponding to a dilution of 1:134) (p < 0.001). Inter-assay variability was determined by testing one HCV genotype 2 sample in 10 consecutive experiments (n = 10), whereas intra-assay variability was evaluated by testing the same sample 10 times (n = 10) in the same experiment, whilst running the dilution series. The intra-assay and inter-assay coefficients of variation (CV) of the log neutralization titers were 6.7% and 12.6%, respectively. Fifty-seven HCV-positive antibodies samples were evaluated using the HCV focus reduction neutralization assay. The genotypes were distributed as follows; for types 1a, 1b, 2, 3, 4 and 5, we studied 11, 11, 11, 12, 10 and 2 samples, respectively. The mean values of the different genotypes is shown in Fig. 3 . and Table 1 . The mean log neutralization titers for genotypes 1a, 2 and 3 are very similar (2.046 ± 0.671 for genotype 1a, 2.128 ± 0.365 for genotype 2 and 2.148 ± 0.478 for genotype 3). The mean average values are lower for genotype 1b (1.747 ± 0.462) and genotype 4 (1.786 ± 0.236). Strikingly, very high heterologous titers were observed for five patients -three infected with HCV genotype 1a and two infected with HCV genotype 3 (see Fig. 3a ). There were too few genotype 5 samples to compare with the other genotypes but the corresponding results nevertheless indicate that the neutralization assay is suitable for this genotype. The two The distribution of the log neutralization titers across all the HCV ELISA and RNA-positive samples as a function of the HCV genotype is shown in Fig. 3b . More than 60% of the neutralizing antibodies titers fell in the range from 1.7 to 2.69 log titers, corresponding to dilutions of 1:50 and 1:500, respectively. Overall, 3.5% of the samples displayed a titer greater than log 3.0 (1:1000) and, conversely, 3.5% displayed a titer below the cut-off value, i.e. log 1.25 (1:10). Thus, of 57 HCV-infected patients, only two did not test positive for neutralizing antibodies in this assay (the titers were 0.960 and 0.932, respectively). The role of neutralizing antibodies during acute and chronic viral infection remains an important question and has generated controversial results. Initially, the presence of neutralizing antibodies was shown to control the HCV load and to contribute to viral eradication in patients capable of clearing the infection [13] . In other studies, the appearance of neutralizing antibodies was delayed and restricted to IgG1 antibodies in patients who develop a chronic infection [2, 21] . The chimpanzee model has been critical for the study of HCV transmission and host immune responses; however, neutralizing antibodies were not detected in some animals that resolved their infection -suggesting a minimal role in viral clearance, as also observed in human studies [14, 15] . Experimentally infected chimpanzees and naturally infected humans can be re-infected with homologous and heterologous HCV strains, suggesting that the humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing [22] [23] [24] . During chronic infection in humans, the presence and/or production of neutralizing antibodies do not suffice for curing the infection but could regulate the spread of the virus. Thus, it can be postulated that during chronic infection, viral mutants can continuously escape the renewed production of neutralizing antibodies. Retroviral pseudoparticles have been used to develop a very interesting tool for measuring neutralizing antibodies in vitro [14] . The assay has demonstrated the presence of HCV-neutralizing antibodies in human sera with relatively high titers (>1:320) and broadly neutralizing activity against different HCV genotypes. However, this model does not represent genuine HCV virions; in particular, the budding of retroviral particles is thought to be very different and may involve a variety of cellular pathways. Characterization of infectious retroviral pseudotype particles bearing HCV glycoproteins have been shown to be very heterogeneous, and so it is possible that these pseudoparticles may not be as relevant as the native HCV virions [25] . The recent development of a cell culture model for HCV enables the production of native HCV virions that can be efficiently propagated in cell culture [17] [18] [19] . This cell culture system has allowed us to develop a neutralization assay for evaluating the level and the proportion of HCVneutralizing antibodies in chronically infected HCV patients. We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26] [27] [28] . The assay relies on the ability of the specific JFH-1 genotype 2 viral strain to replicate and multiply on a Huh-7 human hepatoma cell line in a cell culture model, enabling the rapid detection of viral foci after 72 hours of infection. Moreover, no secondary foci were detectable at this time point. Fixation with paraformaldehyde and permeabilization with Triton X-100 were chosen in order to preserve antigenicity and prevent the cell monolayer from detaching during washes. Development with DAB peroxide substrate made it easy to count specifically coloured viral foci. The viral inoculum size is an important parameter; it has to be low enough to enable good assay sensitivity but high enough to produce a statistically significant number of foci, i.e. allowing the reduction in the number of foci (and thus the effect of neutralization) to be monitored. Thus, 100 FFUs were used as the inoculum in this neutralization assay. In order to test different human samples, we had to take into account the ability of HDL to facilitate HCVcc entry via a mechanism which depends on expression of the scavenger receptor BI [11, 15, 16, 20] . Given HDL's role in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibodies titer; this frees the assay of the risk of non-specific neutralization activity of the serum via the effects of HDL, the complement system and/or serum amyloid A protein (SAA) [29] . The HCV neutralization assay exhibited good reproducibility, for both duplicate assays and independent tests. As expected, the intra-assay coefficient of variation (CV) was lower than the interassay CV. The test also showed good specificity, since there was no interaction with anti-HIV, anti-HBV or heterophile antibodies. Very low titers were found with HCV ELISA and RNA-negative samples, and the assay's cut-off was determined as the mean titer for negative samples plus two standard deviations (1.25 log, corresponding to a dilution of 1:18). Given that only the JFH-1 strain of HCV genotype 2a was available for the assay, we evaluated the neutralization titer of sera from patients chronically infected with other HCV genotypes, i.e. 1, 2, 3, 4 and 5. Most of these sera were detected as positive by the neutralization assay, except for two sera from HCV genotype 1-infected patients. These two samples presented a high specific antibody ratio according to the ELISA but only very low inhibition by neutralization assay (far below the cut-off, in fact). We conclude that either the samples lacked neutralizing antibodies or that any such antibodies that were present did not cross-neutralize with HCV genotype 2a. The sensitivity was 100% -not only for genotype 2 (the genotype of the strain used for the assay) but also for other HCV genotypes (except genotype 1). HCV genotype 5 antibodies were also measured but there were too few samples for accurate testing. Moreover, the positive sera (96.5%) had comparable and significantly high titers (1.99 ± 0.63), whatever the genotype. This finding suggests that most neutralizing antibodies are cross-reactive. Another possibility is that most of the patients had been previously infected by a genotype 2 strain. However, this is unlikely because few genotype 2 strains are circulating in France [30] . As expected for a neutralization test, the assay presented in the present study appeared to be very specific (independently of the genotype) and usable in most circumstances. For most viral infections, neutralization assays such as that described in this study are used as reference assays. Thus, we are confident that as other HCVcc genotypes become available, these assays will replace the pseudoparticle assay in the near future because they are probably more relevant. Our assay is somewhat time-consuming and could be simplified by using one dilution to count the foci; however, this type of "short cut" would make it difficult to extrapolate to the dilution neutralizing 50% of the inoculum. Another approach would consist in using recombinant HCV capable of expressing reporter genes (such as luciferase) in order to use a single dilution and obtain a quantitative result [31] . However, further neutralization studies using other genotypes are needed in order to complete our observations and to char- A simple, specific and reproducible cell culture-based neutralization assay was developed for the determination of neutralizing anti-HCV antibodies in human sera. This test should be an important tool for gauging the relationship between the neutralizing response and viral load kinetics in acutely and chronically infected patients. The Huh-7 human hepatoma cells [32] were grown in Dulbecco's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum. All cell cultures were maintained in 5% CO 2 at 37°C. The plasmid pJFH-1 containing the full-length cDNA of the JFH-1 isolate (which belongs to subtype 2a (GenBank accession no. AB047639)), was a gift from Dr Wakita (Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) and has been described previously [17] . To generate genomic HCV RNA, the plasmid pJFH-1 was linearized at the 3' end of the HCV cDNA and used as a template for in vitro transcription, as described previously [33] . Viral stocks were obtained by harvesting cell culture supernatants and freezing them at -80°C. Virus titration was performed on Huh-7 cells with 6-well microtiter plates (Corning, NY) 72 hours after incubation, by immunostaining the cells with antibodies from a HCV-positive patient serum that had previously been inactivated at 56°C (see the section on the virus neutralization assay). The viral titer was determined in triplicate from the mean number of foci and expressed as focus forming units/mL (FFU/mL). Seventy-seven human serum samples were tested. Collection of the sera was approved by the local Ethics Committee and informed consent had been obtained from the donors. Fifty-seven of these samples were obtained from chronically infected HCV patients. The presence of HCV antibodies was determined and confirmed using two third-generation HCV EIA assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany and Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom). HCV RNA was determined with a qualitative commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France) and HCV genotyping was performed by direct sequencing, as described elsewhere [34] . The genotypes were distributed as follows: 11, 11, 11, 12, 10 and 2 samples of types 1a, 1b, 2, 3, 4 and 5, respectively. A set of 20 anti-HCV-negative serum samples was used to evaluate the assay's specif-icity, including five serum samples with positive hepatitis B virus surface antibody (anti-HBs) status and five sera from Epstein-Barr virus-infected patients that had tested positive for heterophile antibodies. All serum samples had been stored at -80°C upon collection and had not been thawed until the time of assay. Serum immunoglobulins G (IgG) fraction was purified using protein G-Sepharose (GE Healthcare, Orsay, France The HCV focus reduction neutralization assay was performed in 96-well microtiter plates. Serial dilutions of purified IgG (10 μg) ranging from 1:10 to 1:1,280 were established. Each dilution was tested twice. 25 μL of each sample was mixed with 25 μL of virus (100 FFU) in 96well microtiter plates and incubated for 1 hour at 37°C, 5% CO 2 . A volume of 100 μL of Huh-7 cell suspension (10,000 cells/well) in culture medium was added and incubated for 5 hours at 37°C, 5% CO2. After 5 hours, the supernatants were removed and 100 μL of culture medium were added to the monolayers. After 72 hours, the cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Primary antibody (a HCVpositive patient serum inactivated at 56°C) was diluted to 1:500 prior to use and then incubated for 1 h at room temperature. A peroxidase-coupled, Fc-specific anti-human IgG antibody (Sigma, Saint Quentin Fallavier, France) diluted to 1:200 was dispensed onto the cell monolayer and incubated for 30 min at room temperature. The reaction was developed with DAB peroxidase substrate (Sigma, Saint Quentin Fallavier, France) and stopped after 10 min of incubation with distilled water. The number of HCV foci in each dilution was determined. Controls were included in each assay (non-neutralized virus, purified IgG from each patient at a 1:10 dilution). The dilution that neutralized 50% of the virus was calculated by curvilinear regression analysis using XLSTAT 2006 software (Addinsoft SARL, Paris, France) [35] . Each titer was deter-mined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. Titers were expressed as logarithmic values and means ± standard deviation were calculated. Student's t-test was used to compare data between groups. p values below 0.05 were considered to be significant.

What is the long-term risk of chronic hepatitis C infection?

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852297/ SHA: ee8dca216514deeed4c9415bc2ad8a78dc3d9670 Authors: Fournier, Carole; Duverlie, Gilles; François, Catherine; Schnuriger, Aurelie; Dedeurwaerder, Sarah; Brochot, Etienne; Capron, Dominique; Wychowski, Czeslaw; Thibault, Vincent; Castelain, Sandrine Date: 2007-03-30 DOI: 10.1186/1743-422x-4-35 License: cc-by Abstract: BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies. Text: Hepatitis C virus (HCV, a member of the Flaviviridae family) is an enveloped, positive-stranded RNA virus that preferentially replicates in hepatocytes. At least 170 million people worldwide are persistently infected with hepatitis C virus. Chronic HCV infection is associated with a significant risk of progression to cirrhosis and hepatocellular carcinoma [1] . Antiviral therapy with pegylated alpha-interferon and ribavirin (the current best therapeutic regimen) is only successful in about 50% of all treated patients. Better knowledge of the viral and host factors that determine HCV clearance or persistence during the acute stage of infection is needed in order to improve antiviral therapy and to develop efficient vaccines. Studies focusing on innate and cellular immune responses have shown that a sufficiently large HCV inoculum is able to evade, subvert or circumvent the host's defences. At present, the chimpanzee is the only reliable experimental animal model in which the initial post-HCV infection events and the efficacy of vaccine candidates can be evaluated [2] . It has been shown that HCV-specific T-cell immunity is important in the control of HCV infection [3, 4] . Several studies have indicated a role for humoral immunity in the acute stage of HCV infection but this aspect remains poorly characterized. The E1 and E2 glycoproteins are thought to be the viral attachment proteins and thus the main targets for HCV-neutralizing antibodies; identification of protective epitopes conserved across different strains of HCV is therefore a major challenge in vaccine design. A number of antibodies capable of blocking E2 binding to cells or cell receptors have been described, [5] [6] [7] [8] some of which neutralize HCV entry in animal or cellular models [9, 10] . Cell entry has been shown to involve several surface molecules (notably including the tetraspanin CD81 and the SR-BI receptor [11, 12] ), although further studies are needed to better understand how viral entry occurs and how it might be neutralized. Detection of neutralizing antibodies in human blood had been problematical until an efficient and reliable cell culture system for HCV became available. Hence, the development of an in vitro neutralization assay for HCV could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential of passive and active immunization against hepatitis C. Recent studies using an in vitro neutralization assay system (based on infectious retroviral pseudoparticles (HCVpp) bearing HCV envelope glycoproteins) have confirmed that HCV-infected patient sera can indeed neutralize infection [13, 14] . However, it has also been shown that the neutralizing activity of antibodies from HCV-infected patients is attenuated by a factor present in human serum, identified as the highdensity lipoprotein (HDL) fraction [11, 13, 15] . HDL facilitation of HCVpp entry is a post-binding event [16] , sug-gesting that HDLs favour internalization of virions and thus the latter's escape from neutralizing antibodies. Recently, an HCV cell culture model (HCVcc) has been developed [17] [18] [19] , allowing the production of virus particles that can be efficiently propagated in cell culture. Some preliminary neutralization assays have been carried out by these authors. In this study, we describe how we set up a standardized focus reduction neutralization assay based on HCVcc. Focus reduction assays have been widely used to evaluate the neutralizing antibody responses to viruses that can form foci in infected cells. Following the recent development of the HCVcc model, the principle of the focus reduction assay has been applied to HCV-neutralizing antibodies detection. The JFH-1 HCV 2a viral strain was grown on a Huh-7 human hepatoma cell line. After three days of infection and cell permeabilization, detection of the HCV foci was carried out using an inactivated HCVpositive patient serum primary antibody and a peroxidase-coupled, Fc-specific anti-human IgG-antibody. The reaction was revealed with DAB peroxidase substrate. The viral foci were thus stained brown, making them easy to count (see Fig. 1a ). It has been recently shown that the neutralizing activity of HCV antibodies is attenuated by a serum factor associated with the HDL fraction. Hence, HDLs were able to facilitate HCVpp and HCVcc entry via a mechanism which depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function [11, 15, 16, 20] . In view of the role of HDL in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibody titer (see Fig. 1b ). The specificity of the HCV neutralization assay was assessed by testing 20 anti-HCV-ELISA-negative samples, including five positive for hepatitis B virus surface antibodies (anti-HBs) and five positive for heterophile antibodies. All samples tested negative with two commercial anti-HCV antibody detection assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany; Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom) and HCV-RNA-negative with a qualitative, commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France). These anti-HCV-negative samples were compared with 11 samples from patients chronically infected with HCV genotype 2. The neutralization titers of anti-HCV-negative serum samples are shown in Fig. 2 ., with a mean value of 1.083 ± 0.083 (corresponding to a dilution of 1:12). The assay's cut-off (determined as the mean value for negative samples plus two standard deviations) corresponded to a dilution of 1:18. The assay exhibited specificity and sensibility values of 100% and 96.5%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples (data not shown). Conversely, the chronically HCV genotype 2-positive samples displayed strong reactions, with a mean value of 2.128 ± 0.365 (corresponding to a dilution of 1:134) (p < 0.001). Inter-assay variability was determined by testing one HCV genotype 2 sample in 10 consecutive experiments (n = 10), whereas intra-assay variability was evaluated by testing the same sample 10 times (n = 10) in the same experiment, whilst running the dilution series. The intra-assay and inter-assay coefficients of variation (CV) of the log neutralization titers were 6.7% and 12.6%, respectively. Fifty-seven HCV-positive antibodies samples were evaluated using the HCV focus reduction neutralization assay. The genotypes were distributed as follows; for types 1a, 1b, 2, 3, 4 and 5, we studied 11, 11, 11, 12, 10 and 2 samples, respectively. The mean values of the different genotypes is shown in Fig. 3 . and Table 1 . The mean log neutralization titers for genotypes 1a, 2 and 3 are very similar (2.046 ± 0.671 for genotype 1a, 2.128 ± 0.365 for genotype 2 and 2.148 ± 0.478 for genotype 3). The mean average values are lower for genotype 1b (1.747 ± 0.462) and genotype 4 (1.786 ± 0.236). Strikingly, very high heterologous titers were observed for five patients -three infected with HCV genotype 1a and two infected with HCV genotype 3 (see Fig. 3a ). There were too few genotype 5 samples to compare with the other genotypes but the corresponding results nevertheless indicate that the neutralization assay is suitable for this genotype. The two The distribution of the log neutralization titers across all the HCV ELISA and RNA-positive samples as a function of the HCV genotype is shown in Fig. 3b . More than 60% of the neutralizing antibodies titers fell in the range from 1.7 to 2.69 log titers, corresponding to dilutions of 1:50 and 1:500, respectively. Overall, 3.5% of the samples displayed a titer greater than log 3.0 (1:1000) and, conversely, 3.5% displayed a titer below the cut-off value, i.e. log 1.25 (1:10). Thus, of 57 HCV-infected patients, only two did not test positive for neutralizing antibodies in this assay (the titers were 0.960 and 0.932, respectively). The role of neutralizing antibodies during acute and chronic viral infection remains an important question and has generated controversial results. Initially, the presence of neutralizing antibodies was shown to control the HCV load and to contribute to viral eradication in patients capable of clearing the infection [13] . In other studies, the appearance of neutralizing antibodies was delayed and restricted to IgG1 antibodies in patients who develop a chronic infection [2, 21] . The chimpanzee model has been critical for the study of HCV transmission and host immune responses; however, neutralizing antibodies were not detected in some animals that resolved their infection -suggesting a minimal role in viral clearance, as also observed in human studies [14, 15] . Experimentally infected chimpanzees and naturally infected humans can be re-infected with homologous and heterologous HCV strains, suggesting that the humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing [22] [23] [24] . During chronic infection in humans, the presence and/or production of neutralizing antibodies do not suffice for curing the infection but could regulate the spread of the virus. Thus, it can be postulated that during chronic infection, viral mutants can continuously escape the renewed production of neutralizing antibodies. Retroviral pseudoparticles have been used to develop a very interesting tool for measuring neutralizing antibodies in vitro [14] . The assay has demonstrated the presence of HCV-neutralizing antibodies in human sera with relatively high titers (>1:320) and broadly neutralizing activity against different HCV genotypes. However, this model does not represent genuine HCV virions; in particular, the budding of retroviral particles is thought to be very different and may involve a variety of cellular pathways. Characterization of infectious retroviral pseudotype particles bearing HCV glycoproteins have been shown to be very heterogeneous, and so it is possible that these pseudoparticles may not be as relevant as the native HCV virions [25] . The recent development of a cell culture model for HCV enables the production of native HCV virions that can be efficiently propagated in cell culture [17] [18] [19] . This cell culture system has allowed us to develop a neutralization assay for evaluating the level and the proportion of HCVneutralizing antibodies in chronically infected HCV patients. We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26] [27] [28] . The assay relies on the ability of the specific JFH-1 genotype 2 viral strain to replicate and multiply on a Huh-7 human hepatoma cell line in a cell culture model, enabling the rapid detection of viral foci after 72 hours of infection. Moreover, no secondary foci were detectable at this time point. Fixation with paraformaldehyde and permeabilization with Triton X-100 were chosen in order to preserve antigenicity and prevent the cell monolayer from detaching during washes. Development with DAB peroxide substrate made it easy to count specifically coloured viral foci. The viral inoculum size is an important parameter; it has to be low enough to enable good assay sensitivity but high enough to produce a statistically significant number of foci, i.e. allowing the reduction in the number of foci (and thus the effect of neutralization) to be monitored. Thus, 100 FFUs were used as the inoculum in this neutralization assay. In order to test different human samples, we had to take into account the ability of HDL to facilitate HCVcc entry via a mechanism which depends on expression of the scavenger receptor BI [11, 15, 16, 20] . Given HDL's role in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibodies titer; this frees the assay of the risk of non-specific neutralization activity of the serum via the effects of HDL, the complement system and/or serum amyloid A protein (SAA) [29] . The HCV neutralization assay exhibited good reproducibility, for both duplicate assays and independent tests. As expected, the intra-assay coefficient of variation (CV) was lower than the interassay CV. The test also showed good specificity, since there was no interaction with anti-HIV, anti-HBV or heterophile antibodies. Very low titers were found with HCV ELISA and RNA-negative samples, and the assay's cut-off was determined as the mean titer for negative samples plus two standard deviations (1.25 log, corresponding to a dilution of 1:18). Given that only the JFH-1 strain of HCV genotype 2a was available for the assay, we evaluated the neutralization titer of sera from patients chronically infected with other HCV genotypes, i.e. 1, 2, 3, 4 and 5. Most of these sera were detected as positive by the neutralization assay, except for two sera from HCV genotype 1-infected patients. These two samples presented a high specific antibody ratio according to the ELISA but only very low inhibition by neutralization assay (far below the cut-off, in fact). We conclude that either the samples lacked neutralizing antibodies or that any such antibodies that were present did not cross-neutralize with HCV genotype 2a. The sensitivity was 100% -not only for genotype 2 (the genotype of the strain used for the assay) but also for other HCV genotypes (except genotype 1). HCV genotype 5 antibodies were also measured but there were too few samples for accurate testing. Moreover, the positive sera (96.5%) had comparable and significantly high titers (1.99 ± 0.63), whatever the genotype. This finding suggests that most neutralizing antibodies are cross-reactive. Another possibility is that most of the patients had been previously infected by a genotype 2 strain. However, this is unlikely because few genotype 2 strains are circulating in France [30] . As expected for a neutralization test, the assay presented in the present study appeared to be very specific (independently of the genotype) and usable in most circumstances. For most viral infections, neutralization assays such as that described in this study are used as reference assays. Thus, we are confident that as other HCVcc genotypes become available, these assays will replace the pseudoparticle assay in the near future because they are probably more relevant. Our assay is somewhat time-consuming and could be simplified by using one dilution to count the foci; however, this type of "short cut" would make it difficult to extrapolate to the dilution neutralizing 50% of the inoculum. Another approach would consist in using recombinant HCV capable of expressing reporter genes (such as luciferase) in order to use a single dilution and obtain a quantitative result [31] . However, further neutralization studies using other genotypes are needed in order to complete our observations and to char- A simple, specific and reproducible cell culture-based neutralization assay was developed for the determination of neutralizing anti-HCV antibodies in human sera. This test should be an important tool for gauging the relationship between the neutralizing response and viral load kinetics in acutely and chronically infected patients. The Huh-7 human hepatoma cells [32] were grown in Dulbecco's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum. All cell cultures were maintained in 5% CO 2 at 37°C. The plasmid pJFH-1 containing the full-length cDNA of the JFH-1 isolate (which belongs to subtype 2a (GenBank accession no. AB047639)), was a gift from Dr Wakita (Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) and has been described previously [17] . To generate genomic HCV RNA, the plasmid pJFH-1 was linearized at the 3' end of the HCV cDNA and used as a template for in vitro transcription, as described previously [33] . Viral stocks were obtained by harvesting cell culture supernatants and freezing them at -80°C. Virus titration was performed on Huh-7 cells with 6-well microtiter plates (Corning, NY) 72 hours after incubation, by immunostaining the cells with antibodies from a HCV-positive patient serum that had previously been inactivated at 56°C (see the section on the virus neutralization assay). The viral titer was determined in triplicate from the mean number of foci and expressed as focus forming units/mL (FFU/mL). Seventy-seven human serum samples were tested. Collection of the sera was approved by the local Ethics Committee and informed consent had been obtained from the donors. Fifty-seven of these samples were obtained from chronically infected HCV patients. The presence of HCV antibodies was determined and confirmed using two third-generation HCV EIA assays (Axsym ® HCV Version 3.0, Abbott, Wiesbaden, Germany and Vitros ® Anti-HCV reagent pack, Ortho-Clinical Diagnostic, High Wycombe, United Kingdom). HCV RNA was determined with a qualitative commercial assay (Cobas Amplicor HCV test Version 2.0, Roche Diagnostics, Meylan, France) and HCV genotyping was performed by direct sequencing, as described elsewhere [34] . The genotypes were distributed as follows: 11, 11, 11, 12, 10 and 2 samples of types 1a, 1b, 2, 3, 4 and 5, respectively. A set of 20 anti-HCV-negative serum samples was used to evaluate the assay's specif-icity, including five serum samples with positive hepatitis B virus surface antibody (anti-HBs) status and five sera from Epstein-Barr virus-infected patients that had tested positive for heterophile antibodies. All serum samples had been stored at -80°C upon collection and had not been thawed until the time of assay. Serum immunoglobulins G (IgG) fraction was purified using protein G-Sepharose (GE Healthcare, Orsay, France The HCV focus reduction neutralization assay was performed in 96-well microtiter plates. Serial dilutions of purified IgG (10 μg) ranging from 1:10 to 1:1,280 were established. Each dilution was tested twice. 25 μL of each sample was mixed with 25 μL of virus (100 FFU) in 96well microtiter plates and incubated for 1 hour at 37°C, 5% CO 2 . A volume of 100 μL of Huh-7 cell suspension (10,000 cells/well) in culture medium was added and incubated for 5 hours at 37°C, 5% CO2. After 5 hours, the supernatants were removed and 100 μL of culture medium were added to the monolayers. After 72 hours, the cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Primary antibody (a HCVpositive patient serum inactivated at 56°C) was diluted to 1:500 prior to use and then incubated for 1 h at room temperature. A peroxidase-coupled, Fc-specific anti-human IgG antibody (Sigma, Saint Quentin Fallavier, France) diluted to 1:200 was dispensed onto the cell monolayer and incubated for 30 min at room temperature. The reaction was developed with DAB peroxidase substrate (Sigma, Saint Quentin Fallavier, France) and stopped after 10 min of incubation with distilled water. The number of HCV foci in each dilution was determined. Controls were included in each assay (non-neutralized virus, purified IgG from each patient at a 1:10 dilution). The dilution that neutralized 50% of the virus was calculated by curvilinear regression analysis using XLSTAT 2006 software (Addinsoft SARL, Paris, France) [35] . Each titer was deter-mined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. Titers were expressed as logarithmic values and means ± standard deviation were calculated. Student's t-test was used to compare data between groups. p values below 0.05 were considered to be significant.

What antiviral treatments are used for hepatitis C infection?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What is the main cause of death in the neonatal period of calves?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

Where was hepcidin first discovered?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What is hepcidin?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What organ produces hepcidin?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What stimulates the release of hepcidin?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What element does hepcidin play a roles in regulating during metabolism?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

Is hepcidin toxic?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

Why is iron critical to bacteria?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

How does hepcidin work in the duodenum?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

How does hepcidin affect macrophages?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What leads to oxidative stress in the body?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What parameter is used to measure antioxidant levels?

Acute Hemorrhagic Encephalitis Responding to Combined Decompressive Craniectomy, Intravenous Immunoglobulin, and Corticosteroid Therapies: Association with Novel RANBP2 Variant https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857578/ SHA: ef6638accc1ef599ad1aafd47b3a86f2b904cc76 Authors: Alawadhi, Abdulla; Saint-Martin, Christine; Bhanji, Farhan; Srour, Myriam; Atkinson, Jeffrey; Sébire, Guillaume Date: 2018-03-12 DOI: 10.3389/fneur.2018.00130 License: cc-by Abstract: BACKGROUND: Acute hemorrhagic encephalomyelitis (AHEM) is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein (RANBP2) gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone (IVMP) in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease (SCD) presented an acquired demyelinating syndrome (ADS) with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP (30 mg/kg/day). Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins (IVIG). She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant (c.4993A>G, p.Lys1665Glu) was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis (AHEM) or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis (ADEM). AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal (1) (2) (3) . Many treatment options have been used including intravenous (IV) steroids, intravenous immunoglobulins (IVIG), and plasmapheresis (4) . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy (5) (6) (7) (8) (9) . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy (ANE) which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children (10) . We report a new case of AHEM associated to a Ran Binding Protein (RANBP)-2 variant and responsive to combined craniectomy, intravenous methylprednisolone (IVMP), and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease (SCD). A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia [day 0 (D0): refers to the start of the diplopia] 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging (MRI) performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal (Figure 1 ). Brain MR angiography (MRA) showed a normal appearing circle of Willis. The cerebrospinal fluid (CSF) obtained by lumber puncture was normal (WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands). The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate (tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus), and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive (1:1,280) reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies (ANA) were positive (1:160). B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP (30 mg/kg/day) followed by 9 days of oral prednisone (1 mg/kg/day). At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness (D30) with normal alertness. She had normal blood pressure (120/81 mmHg). She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine (D30) she deteriorated with vomiting and reduced level of consciousness (Glasgow Coma Scale dropped from 15 to 8 over 30 min). Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal (Figures 2A-F) . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain (EVD). Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere (Figures 2G-I) . She was started on IVMP (30 mg/kg/ day for 5 days) and IVIG (1 g/kg/day for 2 days). Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions (Figure 3) . Prednisolone was tapered course over 6 weeks. At discharge (D71), she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR (HSV1/2) were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens (SSA-RO, SSB-LA, smith, RNP) were negative. Serum autoimmune antibodies panel (NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI) were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel ( Table 1 ) targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation (NM_006267.4, c.4993A>G, p.Lys1665Glu). This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease (6/117,118 alleles). Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging (SIFT and Align GVGD). Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A (11) . SCD patients are prone to ischemic or hemorrhagic strokes (12) . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case (13) . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients (13) (14) (15) (16) . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient (17) . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis (18) . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia (19) . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes (20, 21) . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context (7, 19) . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy (5) (6) (7) (8) (9) (22) (23) (24) (25) . Autosomal dominant mutations (with incomplete penetrance) in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy (26, 27) . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum (10) . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools (a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid). It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy (25, 26) making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells (28) . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation (2016). This work was supported by grants from Heart and Stroke Foundation of Canada (grant number: G-14-0005756), and Foundation of Stars.

What is Acute hemorrhagic encephalomyelitis?