Influenza viruses A and B are the main pathogens responsible for the onset of epidemics because of their evolving nature. They are RNA viruses that have a high mutation rate and ability to make "drift" changes; however, only influenza A viruses are responsible for pandemics. Worldwide, influenza A viruses are the cause of severe infections in 3-5 million people annually, and these viral infections kills 0.25-0.5 million people annually. 44 As such, influenza outbreaks produce high morbidity and mortality rates with great economic and social impact. 44 Early findings in relation to the most recent influenza pandemic occurred in April 2009 in Mexico and soon spread to other countries. The pandemic was caused by an H1N1 variant, which came from two genetic recombination events. The first occurred in 1998, when an avian virus, an American pig virus, and virus fragments of humans had exchanged genetic materials. The following recombination with a European swine virus strain resulted in the pandemic swine origin influenza virus. 2, 30 In Mexico in recent years, this has caused at least four outbreaks with high mortality rates compared with that presenting in other countries. 6 During the winter of 2017-2018, influenza activity increased in Mexico, and 2,855 cases of influenza and 73 deaths were confirmed by March 02, 2018, of which 46 cases were A(H3N2), 11 cases A(H1N1) pdm09, 10 cases B, and the remaining six cases were not subtyped. 39 Results emanating from different studies have shown that influenza outbreaks are characterized by high severity of symptoms with increased mortality, 6, 8, 13, 25, 32 Several factors have been associated with H1N1 disease severity, such as factors due to the virus (ie, viral pathogenic mutations, resistance to antivirals), factors inherent to host susceptibility (eg, age, sex, race), including physiological immunosuppression or acquired diseases (ie, diabetes, hypertension, obesity, asthma), factors associated with available medical services and public health facilities, and factors arising from the presence of bacterial coinfections. 1, 3, 6, 8, 9, 16, 18, 25, 32, 34, 35, 41 Seasonal and pandemic influenza often have complications arising from bacterial coinfections. Cillóniz et al 12 documented that in H1N1 patients with community-acquired pneumonia, the most frequently isolated bacterial pathogens were Streptococcus pneumoniae (26, 62%) and Pseudomonas aeruginosa (6, 14%) . Staphylococcus aureus was rarely found, and Haemophilus influenzae was not found. 12 During the 1918 pandemic, most deaths had bacterial coinfections. Globally, more than 34% of influenza virus infections needed intensive care among hospitalized patients, from which 0.5% of all cases of influenza corresponded to healthy young individuals and at least 2.5% of total cases the elderly group and those with coinfections harbored the bacteria. 27 Symptoms of influenza cases with bacterial coinfections are similar to those with severe influenza, but the former may have a higher risk of death. Identification of coinfections should be considered in patients with influenza-like illness (ILI) presenting symptoms suggestive of pneumonia, such as dyspnea, tachypnea, and hypoxia, or with evidence of septicemia. 27 Many copathogens are known to be colonizers of the respiratory mucosa, ie, S. pneumoniae, H. influenzae, and Neisseria meningitidis, including the upper and lower respiratory tracts. Distinction between copathogenic colonization and coinfection is critical, because a proven coinfection is clinically correlated with signs of pneumonia and bacterial contagion producing increased acuity or severity of disease. 27 Empirical antiviral treatment should be considered and managed in such critically ill patients. The most commonly isolated bacterial pathogens are those that colonize the nasopharynx, and this complex of virus-bacteria contributes significantly to the pathogenesis of the disease, mainly in periods of endemic influenza. 26, 34 There have been studies reporting copathogens between influenza and other viruses, but few cases have observed that this produced severe complications because of coinfection. 15, 33, 42 Two studies have hypothesized a "viral interference", suggesting that a rhinovirus infection may interfere with the A(H1N1pdm09) influenza, but this is still not fully understood. 24, 33 The precise identification of infectious pathogens responsible for acute respiratory infections, primarily influenza, is a critical factor for proper treatment of the disease and control during outbreaks and for the appropriate use of antibiotics and antivirals. For these reasons, continuation of investigations into the pathogens commonly associated with influenza cases is urgently needed. Here, the presence of bacterial and viral copathogens are identified using clinical samples for the molecular diagnosis by resequencing microarray in study patients with confirmed influenza A(H1N1)pdm09 in Oaxaca, Mexico. We also document an association between influenza A(H1N1pdm 09) and symptoms of disease severity in dead patients with multiple-microbial infection.
The present study involved the collaboration of one government health institution in Mexico that performed the sample collection. Before each examination, each adult who had voluntarily come to the examination point and agreed to participate was informed about the microbiological process of his/her sample, and oral consent was obtained. Parents or guardians provided oral consent on behalf of all under age child participants. The ethical committee of the health secretariat of Mexico approved the use of oral consent, given that the studies were conducted as part of the national H1N1-surveillance program and thus part of a routine public health-monitoring program conducted by the Mexican government. 
Only participants with confirmed H1N1 by qRT-PCR and ILI of any age who had a fever ≥38°C, cough, and headache accompanied by one or more of rhinorrhea, rhinitis, arthralgia, myalgia, prostration, sore throat, chest pain, abdominal pain, or nasal congestion were included in the study. For patients <5 years of age, irritability was substituted for headache. In those >65 years of age, fever was not required as a cardinal symptom.
Individual respiratory clinical samples were collected. These included throat swab, nasopharyngeal swab, bronchoalveolar lavage, and lung biopsy according to each patient's condition. Throat and nasopharyngeal swabs were collected using a rayon or Dacron hyssop in a plastic tube containing 2-3 mL viral transport medium. Bronchoalveolar wash and lung biopsy samples were collected by trained medical staff and placed in plastic bottles containing 15-20 mL viral transport medium. All samples were kept at 2°C-8°C after sampling and during transport to a local molecular biology laboratory and stored at -80°C until testing. 17
Detection of influenza A(H1N1)pdm09 using qRT-PCR RNA extraction Viral RNA was extracted using 140 μL of each clinical sample and of a positive control of influenza A(H1N1) pdm09 (donated by the Laboratory of Molecular Validation and Testing of the Institute for Epidemiological Diagnosis and Reference, Mexico) following the manufacturer's instructions from the QIAamp viral RNA minikit (Qiagen, Venlo, Netherlands). RNA extraction was completed using an automated protocol (QIAcube; Qiagen) and an elution volume of 60 μL. The RNA to be used as template was stored at -80°C until testing.
The protocol included four sets of primers and probes ( 
We followed a US Centers for Disease Control and Prevention protocol (http://www.who.int/csr/resources/ publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf) and used a Fast ABI 7500 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). The total reaction volume was 25 μL, including 5 μL template DNA (4-140 ng/μL), 0.5 μL primer forward (0.4 μM), 0.5 μL reverse primer (0.4 μM), 0.5 μL probe (0.1 μM), 12.5 μL 2× PCR Master Mix, 0.5 μL SuperScript III RT/Platinum Taq Mix, and 5.5 μL sterile molecular biology grade water. The PCR procedure started with one cycle of 50°C for 30 minutes (RT), one cycle of 95°C for 2 minutes (Taq inhibitor activation), followed by 45 cycles of 95°C for 15 seconds and 55°C for 30 seconds, as fluorescence data needed to be collected during the 55°C incubation step. A positive control (10 ng/μL) and a negative control of molecular biology grade water were run in parallel during all experiments. Background fluorescence was considered the correct value of the cycle threshold or cut (C t ). It was thus considered the threshold that went over the background fluorescence for each run in the 
Ramírez-Palacios et al exponential phase of the amplification curve. A cutoff value of C t =37 was corrected accordingly.
With 450 μL of the primary sample, total nucleic acids were recovered following the manufacturer's instructions from the MasterPure DNA-and RNA-isolation kit (Epicentre Biotechnologies, Madison, WI, USA). A final elution volume of 35 μL was stored at -20°C until testing.
Amounts of total nucleic acids were estimated according to the ratio A 260:280 nm using a NanoDrop 2000c (Thermo Fisher Scientific).
With an RPM-Flu 3.1 RT tube containing 4 μL total nucleic acids, RT was performed in order to obtain cDNA. The RT master mix comprised 4 μL 5× buffer for a single chain, 2 μL 0. 
Purification of PCR products was performed following the instructions of the QIAquick PCR purification kit (Qiagen). PCR products of tubes A-D were mixed and purified according to the manufacturer's instructions. A final elution volume of 25 μL was stored at 4°C.
Reagents were used for the fragmentation and labeling of the PCR products for the resequencing GeneChip assay (Affymetrix; Thermo Fisher Scientific). Briefly, in a PCR tube, fragmentation was conducted using 23 μL purified PCR products and 2.6 μL master mix (2.5 μL fragmentation buffer 10× and 0.1 μL fragmentation reagent). PCR cycling conditions were 37°C for 5 minutes and 95°C for 10 minutes. The PCR product was stored at 0°C. Subsequently, a new PCR tube was used to perform the DNA labeling, which contained 25.6 μL fragmented PCR products and 10.4 μL master mix (7.2 μL of TdT buffer 5×, 1.2 μL labeling reagent, and 2 μL TdT 30 UI/μL). PCR cycling conditions were 37°C for 30 minutes and 95°C for 5 minutes. Then, the labeled PCR product was placed for at least 5 minutes in 0°C for hybridization.
The hybridization procedure was carried out for 16 hours at 56°C using a hybridization oven (model 640, Affymetrix). Microarrays were then washed and stained in an Affymetrix Fluidics Station. A computer CEL file containing microarray images was produced by an Affymetrix G7 scanner.
The scanned image of the microarray was analyzed using GSEQ 4.0 software, which produced a CHP file containing the names and the sequences identified in each sample. Sequences were stored in a FASTA file, which was submitted online to retest the sequences on the manufacturer's website (http://www2.gsu.edu/~psyrab/gseq/index.html). Sequences of the pathogens identified in the FASTA file contained the results of the positive and negative controls and an overview of the sequences, including their name, C3 score, homology percentage, and length of the longest continuous sequence. 
Copathogens with H1N1 in Oaxaca, Mexico of each pathogen and symptoms in the 88 patients were also examined per age-group.
The influenza A(H1N1)pdm09 virus was detected and confirmed by qRT-PCR in a total of 88 clinical samples from study patients showing ILI in Oaxaca, Mexico (Table S1) .
To identify associated pathogens in the 88 samples, we used the microarray RPM-Flu 3.1.TessArray. A total of 71 patients presented copathogens with bacteria and/or viruses other than influenza A(H1N1)pdm09 (Table S1) (Figure 1) . A medium number (14.7%) of prevalent bacteria were P. aeruginosa. A few patients (range of prevalence 2.2%-7.9%) harbored other species of bacteria, such as H. parainfluenzae, S. aureus, P. putida, P. mendocina, and N. gonorrhoeae. Less prevalent species of bacteria and viruses (1.1%) found are also summarized in Figure 1 . Table 3 highlights the copathogens identified as colonizers of the respiratory tract and coinfection with pathogens found in the 88 H1N1 patients of Oaxaca.
Of the 22 symptoms identified, the most common were fever in 85 patients (96%), impaired general health in 85 (96%), cough in 84 (95%), headache and myalgia in 75 (85%), rhinorrhea in 72 (82%), odynophagia in 57 (65%), cold in 56 (64%), and nasal congestion in 52 (59%). Most signs and/or symptoms were found among study participants 0.4-30 years old infected with influenza A(H1N1)pdm09 and microbial infection ( Figure 2 ). Six of the 88 patients experienced disease severity and were admitted to the intensive care unit (Table S1 ).
Two of four dead patients harbored three bacteria. One male aged 30 years had S. pneumoniae, H. influenzae, and N. meningitidis. He was treated on an outpatient basis, and (Table S1 ) and died during the period of confinement (puerperium) just after childbirth. The remaining two dead patients harbored two bacteria. One male aged 22 years had S. pneumoniae and N. gonorrhoeae. He was treated on an outpatient basis, presented multiple symptoms, and was asthmatic. One 8-month-old male had H. influenza and S. mitis. The baby presented multiple symptoms, and was also asthmatic.
Mexico has suffered at least four outbreaks produced by the influenza A(H1N1)pdm09 virus, with higher mortality rates than those reported by other countries. 4 Here, we used the standard qRT-PCR protocol to identify the influenza A(H1N1)pdm09 virus in conjunction with a microarray assay for detecting other associated pathogens in positive patients to influenza A(H1N1)pdm09. The microarray assay has been shown to be capable of detecting in a single sample up to 30 viruses and bacteria that may produce ILI. 28 The identification of such pathogens is based on sequence analysis and subsequent search in the GenBank database, which helps to identify specific viral types and species of bacteria present in the sample. 28, 43 There was a significantly high prevalence rate of influenza patients with one and three associated pathogens ( Table 2 ). The bacteria species most frequently identified was S. pneumoniae (39.8% of total individuals with copathogens), which differs somewhat with percentages (7.5%-18.6%) reported elsewhere. 3, 8, 13, 29 H. influenzae (32.9%) and N. meningitidis (25%) also showed high prevalence rates. N. meningitidis is associated with greater severity of illness but requires a process of immunosuppression by patients. 5, 14, 23 Other associated pathogens that presented elevated prevalence rates were S. mitis (23.9%) and P. aeruginosa (14.7%), which coincide with other studies that reported (1%-13.9%) coinfections with those pathogens. 3, 29 However, when comparing the results of this study with previous studies, prudence should be exercised, given that anatomic sites sampled and techniques of detection (and sensitivity and specificity) were somewhat different.
It is noteworthy that the presence of a high number of patients harboring several pathogens producing respiratory infections can be explained by considering the sanitary conditions of a particular country. For example, the prevalence of meningococcal disease varies among countries: 0.3-4 cases per 100,000 people. 36 In addition, there were only three cases where the pandemic virus was found in coinfection with parainfluenza 3 and Coxsackie virus. The results of our study are consistent with other studies that have reported lower rates of viral coinfections with respiratory syncytial virus, coronavirus, influenza B virus, adenovirus, and parainfluenza virus without significant complications or increase in the severity of the disease. 3, 15, 40 Similarly, some associations were found in most patients with certain signs and/or symptoms with influenza A(H1N1)pdm09 alone and with other pathogens (Table 3) . Although this table does not address severity of illness or indicate that treatment of the bacterial infection was necessary, the evolution in the clinical status of the patients was more severe in some patients who interestingly 
Copathogens with H1N1 in Oaxaca, Mexico 
Ramírez-Palacios et al harbored copathogens: six patients of this group (6.8%) were admitted to the intensive care unit, and four (4.5%) died as a consequence of the coinfections. 3, 15, 40 The 30-year-old male could have died as a consequence of infection with N. meningitidis, as influenza could have facilitated meningococcal colonization. 5, 19 The 29-year-old female was in puerperium and presented 11 symptoms, H1N1, and three pathogenic bacteria, such as S. aureus. One study found that the majority of S. aureus isolated in both children and adults were methicillin resistant. 27 As a consequence, she was treated as inpatient in an intensive care unit.
The other two dead patients were asthmatic. They presumably died because adults and children with asthma are more likely to develop pneumonia after getting sick with the flu than people who do not have asthma (https://www.cdc. gov/flu/pdf/freeresources/updated/treating-influenza-2017. pdf). The 22-year-old male harbored S. pneumoniae, which appears to have a synergistic relationship with influenza. 38 This patient should have been treated on an inpatient basis and sent to the intensive care unit, as he was asthmatic and presented 10 different symptoms. However, he was treated on an outpatient basis. The 8-month-old male had eight symptoms and harbored S. mitis, which could have elevated the risk of and exacerbated the influenza infection. 21 A total of 21 of 88 H1N1 patients harbored S. mitis. All H1N1 patients who had complications and those that were submitted to intensive care units were treated with the neuraminidase inhibitor oseltamivir (Tamiflu) within 48 hours of first symptoms of infection. An increase in the number of S. mitis cases could also have hampered the efficacy of the viral neuraminidase inhibitor drug. 21 Of the four H1N1 patients who died, S. mitis was present in three.
Copathogens were found in a significant number (>50%) of influenza cases in Oaxaca. Although some copathogens may be simply asymptomatic colonizing bacteria, other manifested proven coinfection and disease severity in some patients. Given that copathogens were found commonly, this could have an impact on the disease, but more studies need to be conducted, eg, investigating if coinfections between the pandemic virus and other pathogens can be the source of secondary infections when patients are in a status of immunosuppression. Moreover, other factors explain the increase in the severity of symptoms and mortality rate caused by H1N1 in Oaxaca. It has been noted that in areas of Oaxaca, sanitary conditions are not adequate, and this may allow proliferation of the pathogenic agents detected. A monitoring and surveillance system based on molecular diagnostics for all respiratory pathogens should be implemented, as well as strengthening of sanitary measures in the local Oaxaca population, both of which are urgently needed.
Copathogens with H1N1 in Oaxaca, Mexico 