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"PMC7114196"

"25175676"

"Introduction Gastrointestinal and hepatic diseases in dogs are the third most frequent problem reported by owners in United States and Australia ( Freeman et al., 2006 ). Diarrhea represents one of the most frequent disorders in dogs examined at private veterinary practice, with a prevalence of 2.2% ( Lund et al., 1999 ), young dogs under 6 months of age being at a higher risk of diarrhea than adult dogs ( Tupler et al., 2012 ). In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death ( Grellet et al., 2012 ). A great variety of parasites and viruses are described to be enteropathogens during the weaning period in puppies. Giardia duodenalis , Cryptosporidium parvum , Toxocara canis , Cystoisospora ohioensis complex, Cystoisospora canis , canine parvovirus type 2 (CPV2) and canine coronavirus (CCV) are the most prevalent ( Hackett and Lappin, 2003 ). However, as in other species, diarrhea is multifactorial, involving factors intrinsic to the dog (breed size and age), nutritional factors (diet change without transition, food type and quality), together with lifestyle and environmental stressors ( Weber et al., 2002 , Weber et al., 2003 , Sokolow et al., 2005 , Hernot et al., 2006 , Stavisky et al., 2011 ). Most studies on risk factors of diarrhea in young dogs focused on one single pathogen or a group of pathogens without taking into account environmental stressors ( Finlaison, 1995 , Buehl et al., 2006 , Grellet et al., 2012 , Tupler et al., 2012 ). Moreover most of the studies considering multiple enteropathogens infections were performed in shelters, in a context far different from that in breeding kennels ( Sokolow et al., 2005 , Tupler et al., 2012 ). The purpose of this epidemiological study was to determine prevalence of enteropathogens in puppies in breeding kennels and to perform a risk factors analysis for diarrhea during the weaning period including enteropathogens, environment and management procedures."

"Materials and methods Animals and breeding kennels A total of 266 puppies (60 litters) from 29 French breeding kennels were included in this study between May and September 2009 (mean of 9 puppies included per kennel; range: 2–18). Puppies were between 5 and 14 weeks of age (mean: 7.8 weeks of age) ( Fig. 1 ). These breeding kennels were randomly selected from a data base of breeders registered at Alfort Veterinary School for training programs. Only puppies with a normal clinical examination were included (puppies with clinical signs of prostration, dehydration and/or anorexia were excluded of this study). For each kennel, data concerning environmental factors (number of puppies sold per year, and litter size for each puppies included), management of the kennel and puppies (number of meals distributed per day, access to outdoor, vaccination) and puppies’ characteristics (age, breed, sex), were collected. Puppies vaccinated within the preceding 10 days before the visit were not included. Depending on the mean adult body weight of their respective breed, puppies were divided in two groups (small if mean adult body weight < 25 kg; large otherwise). Small breed dogs represented 25.6% (68/266) of the total number of dogs included. Based on the mean number of puppies sold per year (calculated over the last two years and considered as the size of the kennel), kennels were also separated into “small” (i.e. less than 30 puppies sold per year) and large kennels (i.e. more than 30 puppies sold per year). Puppies housed in breeding kennels producing 30 puppies or more per year represented 51.1% (136/266) of the total number dogs included. Puppies were divided into two groups according to the number of meal per day: puppies receiving less than 4 meals per day and puppies receiving 4 meals per days or more. Evaluation of feces consistency For each puppy, fecal consistency was evaluated by a single operator using a 13-point scale, based on the texture and shape of the feces (from liquid to hard and dry) ( Grellet et al., 2012 ). Based on growth rate, thresholds for abnormal feces were previously validated and appeared to vary with breed stature and age ( Grellet et al., 2012 ). Briefly, feces with a score ≤ 5 was classified as abnormal for large breed puppies whatever the age, for small breed puppies, fecal scores ≤6 and ≤7 were classified as abnormal for 4–5 weeks old puppies and for older puppies between 6 and 8 weeks old, respectively. After collection, stools were separated in three samples, one being stored at +4 °C for coproscopy and other frozen (−20 °C) for Giardia intestinalis and Cryptosporidium parvum copro-antigens quantification. A rectal swab was performed for each puppy immediately after stool collection for detection of canine parvovirus type 2 (CPV2) and canine coronavirus (CCV). The swabs were stored at −20 °C until DNA extraction. Intestinal parasites By the standard McMaster flotation technique using saturated magnesium sulphate solution (density: 1.28 g/ml) ( Bauer et al., 2010 ), all eggs and oocysts were identified according to their morphological characteristics under light microscopy by a single operator ( Levine and Ivens, 1965 , Baek et al., 1993 ). Copro-antigens of G. intestinalis and C. parvum were quantified on 100 mg of feces using respectively the ProSpecT-Giardia and the ProSpecT-Cryptosporidium Microplate Assay kit (Remel, France) ( Decock et al., 2003 , Mekaru et al., 2007 , Rimhanen-Finne et al., 2007 ). An optical density value > 0.05 was considered positive according to the manufacturer's instructions. Coronavirus and parvovirus fecal excretions CPV2 and CCV detection were performed by qPCR and qRT-PCR respectively as already described ( Grellet et al., 2012 ). Results from duplicate analyses (mean of two results) were expressed semi-quantitatively as viral load levels. Puppies were defined as excreting CPV2 and CCV for high viral loads over 10 10.3 copies and 10 9.3 copies respectively ( Grellet et al., 2012 ). Data management and statistical analysis Statistical analyses were performed with the SAS version 9.3 software (SAS Institute Inc., Cary, NC, USA). Statistical analysis for prevalence of enteropathogens Number of puppies with fecal positive and negative test results for each enteropathogen was tabled by different factors under study like age of puppies, size of the kennel, breed size, and litter size. Univariate analyses of the putative risk factors for each enteropathogen infection were performed. The significance of the univariate associations was determined using the χ 2 -tests. A P value < 0.05 was considered statistically significant. Statistical analysis for risk factors of abnormal feces Correlation matrix of quantitative and dichotomous variables (excretion of CPV2, CCV, G. intestinalis , C. parvum , T. canis , C. ohioensis complex, and C. canis and number of meal per day, litter size, breeding kennel size) was determined with Kendall's Tau-b measure of correlation coefficient (Proc CORR). These highly correlated variables were defined as predictors in a partial least squares regression (Proc PLS) with fecal consistency as the response variable. The Variable Importance for Projection (VIP) statistic of Wold (1994) was used to assess the contribution of each predictor to the model ( Wold, 1994 ). Only predictors with a VIP value over 0.8 were selected to be included in a new partial least squares regression ( Wold, 1995 ). Variables of the final partial least squares regression with a VIP value over 0.8 were not collinear ( r 2 < 0.10). These variables were subsequently integrated as independent variables and assessed as a fixed effect in a generalized linear mixed model (proc GLIMMIX) with fecal consistency as a binary outcome (logit transformation). As data on puppies were nested within naturally occurring hierarchies (puppies within litter, litters within breeding kennel), litter variable nested within breeding kennel, written as litter (breeding kennel), was defined as a random term. The respective influence of litter and breeding kennel as random effects was also determined."

"Results Prevalence of enteropathogens 77.1% (205/266) of the puppies were infected by at least one enteropathogen with 29.3% of them excreting 3 pathogens or more ( Table 1 , Table 2 ). Seven different viruses and parasites were identified. 14.7% of puppies (39/266) were infected by CPV2, 20.3% (54/266) by CCV, 41% (109/266) by Giardia sp., 25.9% (69/266) by C. parvum , 25.6% (68/266) by C. ohioensis complex, 22.2% (59/266) by T. canis , and 13.2% (35/266) by C. canis . All enteropathogens except T. canis presented a significantly higher prevalence in large breeding kennels. Puppies between 5 and 8 weeks of age presented a significantly higher prevalence of CPV2 and C. ohioensis complex and a lower prevalence of CCV and G. duodenalis than puppies between 9 and 14 weeks of age ( Table 3 ). Risk factors of abnormal feces Sixty six out of 266 feces evaluated (24.8%) were classified as abnormal ( Fig. 2 ). In the initial partial least squares regression CPV2, C. canis , G. intestinalis and the number of meal per day presented a VIP over 0.8. These four factors were included in a new partial least squares regression. CPV2 and number of meal per day were two factors keeping a significant impact on the incidence of abnormal feces with a VIP over 0.8 (VIP = 1.7 and VIP = 1.0 respectively). In the final model only fecal excretion of CPV2 increased risk of weaning diarrhea ( P = 0.003, odds ratio = 5; confidence interval 95%: 1.7–14.7). 61.5% (24/39) of puppies infected by CPV2 presented abnormal feces compared to 15.2% (42/277) of puppies not infected by CPV2. A global significant effect of litter and breeding kennel was observed, with a significant effect of the litter level ( P < 0.001), and no significant effect of the breeding kennel level ( P = 0.101)."

"Discussion The present study represents the first investigation of the prevalence of weaning diarrhea in puppies living in a breeding kennel. Prevalence of this clinical sign, affecting both growth and survival was high with 24.8% of puppies between 5 and 14 weeks concerned. Among the seven different enteropathogens (2 viruses and 5 parasites) tested in this study, 77.1% of puppies were infected by at least one virus or parasite, and 55.3% carried multiple organisms. Prevalence of parasites was higher than the prevalence of viruses (74.4% vs 34.6%). This high prevalence of multiple infections is in accordance with a previous study on dogs entering animal shelters in which 55% of them presented multiple digestive infections ( Tupler et al., 2012 ). In our study, 14.7% of puppies were excreting CPV2, 20.3% by CCV. However, only 0.4% of puppies presented a mixed infection by these two viruses. Prevalence of these viruses depends on age, lifestyle and health status of dogs. Serological and virological investigations demonstrated that CCV and CPV2 are highly prevalent in kennels and animal shelters compared to single owned dogs ( Rimmelzwaan et al., 1991 , Tennant et al., 1993 , Bandai et al., 1999 , Naylor et al., 2001b , Schulz et al., 2008 ). Moreover a higher prevalence of CCV and CPV2 was described in young animals under 6 months of age compared to adult dogs ( Sakulwira et al., 2003 , Gates and Nolan, 2009a , Gates and Nolan, 2009b , Epe et al., 2010 ). In addition to the wide distribution and contagiosity of these viruses, the methods used for detection of these viruses can also contribute to this high prevalence. PCR assays have been proven to be up to 4 × 10 4 times more sensitive than electronic microscopy and virus isolation for detection of CCV ( Naylor et al., 2001a ). This method is able to detect virus in the feces of low-grade shedding animals below 10 6 particles per gram of unprocessed feces, which is considered the detection limit for electronic microscopy. In our study, a higher prevalence of CPV2, CCV was observed in breeding kennels producing 30 puppies per year or more. The higher prevalence in this sub-population could be linked to the contagiousness of these pathogens and their stability in the environment ( Terpstra et al., 2007 , Eterpi et al., 2010 ). The close contact between animals and the density of puppies could promote the environmental contamination and subsequently the spread of the infection. 17.9% of puppies in our study were found excreting a high load of CPV2. This virus is well described as inducing hemorrhagic diarrhea associated with vomiting, anorexia, dehydration and depression ( Meunier et al., 1985 , Prittie, 2004 ). However, in our study, CPV2 also increased risk of weaning diarrhea but without systemic signs, as already described, and 12.5% of dogs without gastrointestinal disease excreted this virus ( Grellet et al., 2012 ). Thus our study demonstrated that puppies can excrete high viral loads of CPV2 without any systemic sign. This observation is in accordance with one previous study in which fecal excretion was also quantified by PCR ( Schmitz et al., 2009 ). However studies using less sensitive methods (fecal antibody-based antigen tests, immune-electron microscopy) did not observed this healthy carrier status ( Hackett and Lappin, 2003 , Desario et al., 2005 , Sokolow et al., 2005 , Schulz et al., 2008 , Schmitz et al., 2009 ). The lack of systemic clinical signs on these puppies could be linked either to an efficient systemic immunity or to local intestinal immunity ( Rice et al., 1982 , Macartney et al., 1988 ). Fecal IgA, endogenous or provided by milk, protect intestinal mucosa by inhibiting the adherence of pathogens, thereby preventing adhesion of these pathogens. Canine milk was found rich in IgA ( Heddle and Rowley, 1975 ) with high levels of CPV2 antibodies ( Decaro et al., 2004 ). These antibodies, repeatedly ingested in large quantities by the puppies during the first weeks of life, could provide some protection to the intestinal mucosa against CPV2 deleterious effects decreasing the systemic clinical signs associated to CPV2 infection (septicaemia, dehydration). Nevertheless these clinically healthy animals probably represent major sources of virus for other animals and for the environmental contamination. Interestingly, in our study, young puppies (between 5 and 8 weeks of age) presented a higher infection rate by CPV2 than older ones. This result highlights the interest of vaccination before 8 weeks of age in breeding kennels to limit CPV2 spreading. The interference with maternally derived antibodies considered as one of the most important causes of immunization failure in puppies ( Macartney et al., 1988 ), can be overcome by the use of high titer CPV2 vaccines ( De Cramer et al., 2010 ). Other infectious agents tested were not associated with weaning diarrhea. In our study, 20.3% of puppies were infected by CCV, but this virus was not identified as a risk factor of abnormal feces. Implications of CCV in acute dog diarrhea are controversial. No relation between coronavirus and diarrhea was observed in different studies, with more healthy dogs infected by this virus than dogs with diarrhea in some of these studies ( Sokolow et al., 2005 , Schulz et al., 2008 , Tupler et al., 2012 ). However, CCV was also described as a virus inducing severe gastroenteritis, lethal in some cases ( Evermann et al., 2005 , Buonavoglia et al., 2006 , Decaro et al., 2008 , Decaro et al., 2009 ). These variations in clinical signs could be linked to variations in pathogenicity between strains ( Escutenaire et al., 2007 ), to the age of infected dogs ( Decaro et al., 2009 ), to the number of genotypes infecting puppies simultaneously ( Decaro et al., 2005 ) or to the association of the coronavirus with other enteropathogens ( Appel, 1988 ). Neither C. Ohioensis complex nor C. canis were associated with weaning diarrhea in our study. Impact of these parasites on weaning diarrhea is still controversial (Buehl, 2006). This difference of clinical signs observed between studies may be explained by differences in the age of infected dogs, the environmental conditions and the virulence of species."

"Conclusion Based on this study, CPV2 infection was the major risk factors of weaning diarrhea. Some central strategies can be suggested like a targeted sanitary and medical prophylaxis against CPV2, particularly in large breeding kennels."

"Diarrhea represents one of the most frequent disorders in dogs. In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death. Prevention of diarrhea in puppies requires a global approach encompassing enteropathogens, environment and management practices especially when housed in groups. The purpose of this study was to determine prevalence of enteropathogens in puppies in breeding kennels and to identify risk factors of diarrhea. Two hundred and sixty six puppies (between 5 and 14 weeks of age) from 29 French breeding kennels were included. For each kennel, data about environment, management of the kennel and puppies’ characteristics (age, sex and breed) were collected. For each puppy, fecal consistency and fecal excretion of enteropathogens (viruses and parasites) was evaluated. At least one enteropathogen was identified in 77.1% of puppies and 24.8% of puppies presented abnormal feces. The main risk factor of weaning diarrhea was fecal excretion of canine parvovirus type 2 (odds ratio = 5; confidence interval 95%: 1.7–14.7). A targeted sanitary and medical prophylaxis against canine parvovirus type 2 should be implemented to decrease risk of weaning diarrhea. Keywords"

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"NO-CC CODE"

"no"

"2023-06-06 23:35:26"

"Prev Vet Med. 2014 Nov 1; 117(1):260-265"

"oa_package/fe/03/PMC7114196.tar.gz"

"PMC7128992"

"31400641"

"Introduction Lung transplantation is an established treatment option for selected patients with end-stage lung disease. Chronic lung allograft dysfunction (CLAD) and infections are the main factors limiting long-term survival in lung transplant recipients (LTRs) [1] . Acute cellular rejection (ACR) is a potential risk factor for the development of CLAD [2] , [3] , [4] , [5] . During the first postoperative year, ACR affects 28% of LTRs at least once, necessitating treatment with steroid augmentation [1] , [6] . Symptoms of ACR are nonspecific, including dyspnea, cough, sputum production, fever and/or hypoxia [7] , [8] . Non-invasive tests like pulmonary function testing and chest imaging are useful indicators for potential complications, but have no discriminatory value between ACR and infection [9] . For these reasons, transbronchial biopsies (TBB) remain the gold standard for the diagnosis of ACR [10] . However, TBB are invasive and bear potential risks such as pneumothorax or bleeding [11] , [12] . Moreover, TBB are prone to sampling error and inter-observer variability [13] , [14] , [15] . The clinical and prognostic role of grade A1 ACR remains unclear. Depending on clinical management guidelines and practice standards at different transplant centers, a finding of grade A1 ACR might be ignored, prompt repeat biopsy or might result in augmented immunosuppression [11] , [16] , [17] . Immense work in multiple laboratories worldwide is currently under way to determine the potential role of cytokines in diagnosis, treatment and monitoring disease progression in various fields such as heart failure, neuro-degeneration and gastrointestinal diseases. Cytokine production by BAL T lymphocytes and mast cells has been shown to be part of pro- and anti-inflammatory processes leading to airflow limitation and exacerbations of obstructive lung disease [18] , [19] . Cytokines are furthermore implicated in rejection after organ transplantation and induction of fibrotic pathways [20] . ACR is driven by T cell recognition of foreign major histocompatibility complexes [8] , [21] . Cytokines play a key role in this process by stimulating proliferation, chemotaxis and activation of cytotoxic T lymphocytes, neutrophils and alveolar macrophages AM [22] , [23] , [24] . We have recently reviewed the potential role of surrogate markers such as cytology and cytokines in bronchoalveolar lavage (BAL) and plasma samples [25] , [26] . Analyzing cytokines in BAL may provide information on allograft status, a potentially useful diagnostic tool. Advances in detection of biomarkers are urgently needed to identify ACR and reliably predict increased risk for the development of CLAD [27] . In this retrospective single-center study, we analyzed a panel of pro-inflammatory cytokines, including interleukin (IL-)6, IL-8, interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in a large cohort of LTRs. The aim of this study was to correlate cytokine levels in BAL fluid of surveillance bronchoscopies with the development of complications including ACR and infection during the first year following lung transplantation. Such correlation might provide a feasible and specific diagnostic potentially allowing early recognition and subsequent targeted treatment of complications in LTRs."

"Patients and methods Study population Starting in 1998, the four cytokines IL-6, IL-8, IFN-γ and TNF-α were analyzed in BAL fluid of LTRs during surveillance bronchoscopies for future research purposes. We enrolled all patients whose medical records included BAL cytokine analyses and concomitantly obtained TBB as well as microbiologic studies. This study was approved by the Cantonal Ethics Committee of Zurich (KEK-ZH number 2016-02148). Immunosuppression protocol and prophylaxis regimen In general, all primary LTRs at University Hospital Zurich receive induction therapy (antithymocyte globulin or basiliximab) and life-long triple immunosuppressive therapy [28] . At our center, cyclosporine A, tapered dose prednisone, and azathioprine or mycophenolate mofetil (since 1999) are used [29] . Anti-infective prophylaxis is used as previously described [30] , [31] . All patients classified as cytomegalovirus (CMV) intermediate-risk or high-risk received prophylaxis with valganciclovir [32] , [33] . In case of CLAD, macrolides are given for immunomodulation [29] . Predictors At regular intervals BAL fluid and TBB were obtained during routine surveillance bronchoscopies during the first year after transplantation (at one, two, three, four, six and 12 months following lung transplantation) [30] . Levels of C-reactive protein (CRP) were routinely measured for all patients before surveillance bronchoscopies. Patients did not undergo surveillance bronchoscopy if clinically unstable, showing clinical signs of infection or infection-associated lung allograft dysfunction or if elevated inflammatory markers in the laboratory. Bronchoscopies performed for specific clinical indications or events were excluded from the study. Lavage was performed in a sub-segmental bronchus of either the lingula or the middle lobe using three to four 50 ml aliquots of 0.9% saline solution [34] . Five to eight TBB were taken from the lower lobe. After fixation in 4% formaldehyde solution and serial section biopsy specimens were stained with hematoxylin and eosin, elastic Van Gieson and Grocott. BAL fluid cytokine concentrations of IL-6, IL-8, IFN-γ and TNF-α were determined by University Hospital Zurich Immunology Laboratory using commercially available, validated quantitative sandwich enzyme immunoassays. The immunoassays use microplates pre-coated with polyclonal antibodies specific for the cytokine and enzyme-linked polyclonal antibodies for their detection in a blinded fashion (R&D Systems; Minneapolis, MN, USA). ELISA kits included Quantikine® ELISA Human IL-6 Immunoassay (catalog # D6050), Quantikine® ELISA Human CXCL8/IL-8 Immunoassay (catalog # D8000C), Quantikine® ELISA Human IFN-γ Immunoassay (catalog # DIF50) and Quantikine® HS ELISA Human TNF-α Immunoassay (catalog # HSTA00D). All cytokines were measured according to the manufacturer’s instruction. The absorbance was measured in an enzyme-linked immunosorbent assay (ELISA) reader (Dynex Opsys MRTM Microplate Reader) at 450/630 nm. The respective cytokine concentration was determined by interpolation from standard curves and expressed as pg/ml. Sensitivity of the assays was 0.7 pg/ml for IL-6, 3.5 pg/ml for IL-8, 8 pg/ml for IFN-γ and 0.1 pg/ml for TNF-α. BAL fluid cytokine concentrations were measured once a week. BAL fluid cytokine samples were stored at 4 °C until processed, for a maximum of one week. Starting in 2013, samples were centrifuged for 10 min at a speed of 2370 g at 4 °C immediately prior to the measurements. The supernatant was removed from the pellet after centrifugation. In summary, 50 μl/well (TNF-α) or 100 μl/well (IFN-γ, IL-6, IL-8) of Assay Diluent were added to the well of the cytokine microplates, then 50 μl/well (IL-8), 100 μl/well (IFN-γ, IL-6) or 200 μl/well (TNF-α) of the respective BAL sample, standards and controls were added as suggested by the manufacturer and incubated for 2 h (IFN-γ, IL-6, IL-8) or 3 h (TNF-α). Plates were washed four times (IL-6), five times (IFN-γ, IL-8) or six times (TNF-α). Removing excess liquid and washing thoroughly is essential; hence, plates with IFN- γ and IL-8 were washed once more than suggested by the provider. Then, 100 μl/well (IL-8) or 200 μl/well (IFN-γ, IL-6, TNF-α) of the respective Conjugate was added and incubated at room temperature for one hour (IL-8) or two hours (IFN-γ, IL-6, TNF-α). Plates were washed four times (IL-6), five times (IFN-γ, IL-8) or six times (TNF-α). Only in the case of TNF- α 50 μl/well of Amplifying Solution had to be added before the addition of the Substrate Solution and incubated at room temperature for 30 min. Then, 50 μl/well (TNF-α) or 200 μl/well (IFN-γ, IL-6, IL-8) of the respective Substrate Solutions were added and incubated at room temperature for 30 min (IFN-γ, IL-6, IL-8) or 60 min (TNF-α). 50 μl/well of Stop Solution was added (IFN-γ, IL-6, IL-8, TNF-α). Plates were read at OD 450/630 nm within 30 min. ACR and microbial detection ACR was assessed and graded in the TBB specimens by experienced pathologists using standard International Society for Heart and Lung Transplantation (ISHLT) nomenclature [35] , [36] . Both Grade A ACR and grade B ACR were considered. Episodes of clinically suspected ACR, antibody-mediated rejection (AMR) and CLAD were not included in the analysis. Specimens were classified as infected if BAL fluid microbiologic studies identified bacteria, viral pathogens, fungi or mycobacteria. Bacterial cultures were considered positive if cultures showed growth greater than 100000 viable organisms per ml, excluding oral flora. PCR was used for detection of respiratory viruses (adenovirus, bocavirus, coronavirus, enterovirus, influenza A, influenza B, metapneumovirus, parainfluenzavirus, parechovirus, rhinovirus and RSV). CMV culture results were not included in the analysis. For this study we did not include the clinical presentation, radiologic findings, macroscopic appearance of BAL fluid and concomitant antibiotic, antiviral or antifungal treatment. Bronchoscopy samples were classified as “no pathologic process” if both TBB and BAL fluid microbiologic studies did not show a pathologic process, irrespective of clinical or radiologic presentations. Statistical analysis Statistical analysis was performed in R (version 3.4.1; R Foundation for Statistical Computing, Vienna, Austria) using “pROC”, “broom”, “lmerTest” and “survival” libraries. Baseline recipient characteristics were expressed as mean (standard deviation) or as median (interquartile range) for continuous variables and as frequency (percentage) for categorical variables. Cytokine concentrations were log transformed prior to analysis to ensure normal distribution. Concentrations too low to detect by ELISA assay were assigned a value of 0.01 pg/ml. Boxplots were generated using default settings in the R graphics library, with whiskers at the default of 1.5 times the interquartile range. Continuous variables were compared using the Mann-Whitney- U test or Kruskal-Wallis test. Categorical variables were compared using Fisher’s exact test. In a multivariable analysis, we controlled for patient characteristics frequently associated with rejection. These included age, underlying lung disease and type of infection (bacterial, viral or fungal). ROC curves for all four cytokines were calculated using the “pROC” package [37] . Overall survival of LTRs was assessed using the Kaplan-Meier method and compared using the log-rank test, with censoring at 31 December 2016. Because the ACR status was assessed after transplantation, we considered it to be a time-varying co-variate in this part of the analysis and had to reshape the data accordingly."

"Results Study population During the study period from February 1998 to November 2016, 425 subjects underwent lung transplantation; 106 subjects were excluded because no cytokine data was recorded in the patient health record system and/or no bronchoscopies were performed. In the remaining 319 subjects, 747 BAL fluid samples were analyzed and compared with TBB specimens obtained during the same bronchoscopy. Median number of TBB and BAL samples per patient was 3 (IQR 1–4). Median time to first TBB sample was 43 (IQR 29–83) days. Patient characteristics are provided in Table 1 . Compared with ISHLT Thoracic Transplant Registry data, our study population included a greater proportion of patients receiving transplantation for CF (33.5% vs. 22.9%) and pulmonary fibrosis (29.1% vs. 24.7%) [1] . Conversely, the patients included in the study received slightly fewer transplants for chronic obstructive pulmonary disease (COPD) (31.3% vs. 31.8%) and pulmonary hypertension (5.3% vs. 6%) [1] . Overall 31.4% of the total 319 patients experienced at least one episode of ACR during the first year post transplantation (vs. 28% ISHLT) [1] . Discrimination between specific groups Bronchoscopy results were grouped based on microbiological and pathological analyses. Of the 747 bronchoscopy specimens, 214 (28.65%) showed “no pathologic process”. Of the remaining samples, 69 (9.24%) showed ACR, 358 (47.93%) infection and 106 (14.19%) “combined ACR and infection”. Table 2 provides an overview of the cytokine concentrations in the different groups. Fig. 1 shows levels of log(IL-6) among the four subgroups; no significant difference was observed. IFN-γ, IL-8 and TNF-α also did not show significant differences by specific group. As shown in Fig. 2 , none of these cytokines had an optimal cut-off to diagnose “combined ACR and infection” or isolated episodes of ACR. Depending on the guidelines of different transplant centers, ACR may only be treated in >A1 ACR episodes. Levels of log(IL-6) during ACR (median 0.37 vs. 0.21) did not differ from “combined ACR and infection” (median 0.67 vs. 0.51), when ACR was defined ≥A2 as opposed to ≥A1 ACR (p = 0.22). No differences were observed in the levels of log(IL-8), log(IFN-γ) and log(TNF-α) (p = 0.21, p = 0.52, p = 0.21, respectively). Factors influencing cytokine pattern Using a linear mixed effects model, we found no significant differences in the cytokine levels by specific group for any of the cytokines. After adjusting for age, underlying disease leading to lung transplantation and type of infection at the time of surveillance bronchoscopy, IL-8 showed significantly lower log(IL-8) levels in patients with infection (2.14) than in patients with “no pathologic process” (2.41, p = 0.02). Also, log(IFN-γ) was lower in patients with ACR only (-1.07), than with “no pathologic process” (−0.77, p = 0.05). log(IL-6) (ACR only 0.39, p = 0.76; infection only 0.19, p = 0.27; “combined ACR and infection” 0.37, p = 0.91, “no pathologic process” 0.35) and log(TNF-α) (ACR only −0.98, p = 0.86; infection only −0.88, p = 0.40; “combined ACR and infection” −0.93, p = 0.65, “no pathologic process” −1.00) did not vary in a significant manner by specific group. Number of events ACR was detected in 175 (23.43%) of all TBB samples (91 showing A1 ACR). 59 patients (18.5%) experienced one event of ACR during the first year after transplantation, 22 patients (7%) experienced two events, and 19 patients (6%) experienced more than two events according to surveillance bronchoscopies. Using a generalized linear mixed model ACR decreased slightly in the first year, with odds ratio of 0.915 per month (p = 0.02). No differences in the rates of ACR event were observed by type of pathogen detected during surveillance bronchoscopies (bacterial infection, p = 0.95; viral infection, p = 0.20; fungal infection, p = 0.79). Survival Among patients undergoing surveillance bronchoscopies with TBB, eight died within the first year after transplantation. Median 1-year conditional survival was 8.6 years for patients without ACR and 7.9 years for patients with a minimum one biopsy-proven ACR in the first year after transplantation, respectively. As shown in Fig. 3 , the 5-year survival rate was similar between patients without (70%) or with episodes of ACR (69%) and higher compared to ISHLT Registry data (57% in era 2009 – June 2015) [1] . There were no significant differences in overall survival between patients with and without ACR (hazard ratio 1.18, 95%-confidence interval 0.69–2.02, p = 0.54), and among patients with ACR, between grade A1 ACR and grade > A1 ACR (hazard ratio 1.00, 95%-confidence interval 0.58–1.71, p = 1.00). There was no correlation with ACR events per patient and survival (p = 0.96)."

"Discussion In this observational study we could not detect a relevant role of IL-6, IL-8, INF-γ and TNF-α in BAL fluid samples of LTRs to identify complications including ACR, infection or both. After adjustment for age, underlying disease leading to lung transplantation and type of infection at time of surveillance bronchoscopy minor differences in the cytokine levels were observed. These data show that the pattern of BAL cytokines is of minor value as a diagnostic marker in LTRs. As reported before [26] data on the role of IL-6 in ACR is conflicting. Whereas some experimental and clinical data showed a significant increase in IL-6 in ACR [22] , [38] , other studies found no significant association at all [39] , [40] . Despite the high number of samples no correlation was found in our analyses. IL-8 was significantly lower during infection than in LTRs with “no pathologic process” after adjustment for age, underlying disease and type of infection. No correlation was found between IL-8 and ACR, which is along the line with most previous studies [22] , [41] , [42] . While IL-8 has been linked with the development of CLAD, its role in detecting ACR seems negligible according to our data [43] , [44] . Levels of IFN-γ were significantly lower during ACR only after correction for age, underlying disease and type of infection. However, in the light of the results from previous studies, these findings should be interpreted with caution [39] , [45] , [46] , [47] . Levels of TNF-α did not correlate with the four prespecified groups. Accordingly, previous studies have suggested no correlation between TNF-α and ACR [22] , [39] , [47] . Of technical note, ELISA is not the best method to detect TNF-α since it is limited in the detection of cytokines that are active in the membrane bound form [48] , [49] . Survival did not differ between patients with ACR and “no ACR” and no difference was seen in patients with multiple ACR events. However, we only included data obtained from surveillance bronchoscopies without clinical signs of allograft dysfunction. Patients are followed up every 1–2 weeks within the first six months following transplantation and 2–4 weeks thereafter just at our center due to the limited size of Switzerland. Any clinical signs of infection or allograft rejection prompt immediate treatment. In addition, patients with multiple ACR events and early signs of CLAD are treated with extracorporeal photophoresis, potentially explaining the higher survival rate compared to the data from the ISHLT registry. In summary, no correlation was found between the cytokines studied here and survival of patients suggesting that these cytokines are not suitable markers for monitoring. This study has several limitations: First, this is a retrospective study. Thus, even though we planned surveillance bronchoscopies at regular intervals, a selection bias can’t be excluded and patients with clinically relevant allograft dysfunction or infection did not undergo surveillance bronchoscopies. This might explain why we derived data from 319 patients, missing the potential of 425 subjects. Also, while we obtained total cell count in BAL samples we did not include this information in our analysis. An increase in total cell count is non-specific after lung transplantation and has also been found in periods with no infection or rejection [50] . Second, we restricted cytokine analysis to a small number of cytokines. When this study was initiated in 1998, these were the only commercially available and validated kits used at our center. All four cytokines had been associated with rejection in previous studies [26] . Also, we did not perform immunologic analyses reflecting the innate immune system such as alpha defensins or matrix metalloproteinases. Further, combining BAL cytokine and cytology levels might contribute to a composite score, increasing diagnostic accuracy for BAL to diagnose ACR. However, evaluating such a score was not part of the study. Third, due to the fact, that AMR was an ill-defined condition before the publication of the 2016 ISHLT Consensus Report [51] we did not include patients with AMR in our study. Fourth, we did not perform analyses to associate cytokines with CLAD development [52] , [53] . The diagnosis of CLAD is an indication to treat patients with an immunomodulatory macrolide. As such, even if the absolute number of patients receiving macrolides at our institution during the first year after lung transplantation is low, a bias of such treatment for cytokine distribution in BAL cannot be excluded [54] . Fifth, statistical methods were not used to adjust for storage time of the BAL samples, number of analyses per patient and date of collection after transplantation. Sixth, infection was defined based on microbiological findings in BAL fluid only and did not take into account clinical symptoms, imaging studies, macroscopic appearance of BAL fluid and concomitant antibiotic, antiviral or antifungal medication. Detecting infection and differentiating infection from ACR based on clinical symptoms may be difficult in LTRs [33] , [55] . Thus, in patients on multiple immunosuppressive drugs the mere presence of pathogens in BAL fluid in the absence of clinical deterioration may fulfill criteria of infection and prompt anti-infective treatment. Indeed, at our center, all pathogenic findings in BAL fluid except for oral flora and candida species prompted anti-infective treatment [30] , [31] . Some aspects of our immunological analysis warrant further discussion. We did not centrifuge BAL specimens before 2013. In a recent internal review conducted by our Immunology Lab comparing cytokine concentrations in BAL samples with and without prior centrifugation, no difference was seen in the majority of samples (unpublished data). Further, we measured BAL cytokine levels once a week and therefore not necessarily on the day they were obtained. Finally, BAL fluid samples were stored at 4 °C until processed, at a maximum of one week. An internal analysis performed in our Immunology Lab in 2012 showed a difference in cytokine concentration <20% between BAL samples stored at 4 °C and samples stored at −20 °C immediately after arrival until processing one week later. There is a need of standardization in BAL technique in LTRs. The ISHLT has recently established a Working Group to address this issue as far as the BAL procedure is concerned, the quotient of instilled volume and aspirated volume should be calculated and provided. This facilitates comparability of cellular and protein concentrations between studies. This would be particularly important for cytokines, as their concentrations are typically low. To further correct for dilution factors of BAL fluid, the urea method has been described, whereby the measured BAL fluid cytokine concentration is adjusted to a urea plasma/BAL coefficient [56] . We did not apply this method in our study to normalize the data. Levy and colleagues recently showed that sequential BAL samples reflect distinct pulmonary compartments [57] . This might have implications for future research. Along this line, centers should agree on a standardized cytokine detection method as comparability between methods is low [48] . ELISA has been a reliable method at our center. However, it is costly and requires strict adherence to time protocols. FACS or Luminex® assays may be alternatives for selected cytokines, yet these methods have yielded less reliable results in our laboratory. Also, numerous potential confounders related to medication (dosage of immunosuppression, anti-infective prophylaxis and treatment, additional individual medication) and immunological analysis make interpretation of cytokine data in LTRs challenging. In the field of rheumatology attempts have been made to harmonize autoantibody nomenclature, thereby optimizing antinuclear antibody usage [58] . Based on the data shown here, the identification of a single biomarker or a profile of different biomarkers is unlikely to provide conclusions on lung allograft function. Whether computed algorithms and precision medicine might help to translate this complex data into the clinical setting is unclear at the moment. In our opinion, this can only be achieved in a collaborative approach by using standardized and validated methods in large prospective multi-center cohort studies. This effort is crucial, however, to optimize survival and quality of life for LTRs [27] . In summary, this is one of the largest retrospective studies to analyze BAL fluid cytokine profiles in LTRs. The cytokines investigated here have no role to diagnose complications after lung transplantation. It is unlikely that further attempts to study these BAL fluid cytokines in the context of ACR, infection or both in LTRs will add novel valuable insights."

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"Early diagnosis and treatment of acute cellular rejection (ACR) may improve long-term outcome for lung transplant recipients (LTRs). Cytokines have become valuable diagnostic tools in many medical fields. The role of bronchoalveolar lavage (BAL) cytokines is of unknown value to diagnose ACR and distinguish rejection from infection. We hypothesized that distinct cytokine patterns obtained by surveillance bronchoscopies during the first year after transplantation are associated with ACR and microbiologic findings. We retrospectively analyzed data from 319 patients undergoing lung transplantation at University Hospital Zurich from 1998 to 2016. We compared levels of IL-6, IL-8, IFN-γ and TNF-α in 747 BAL samples with transbronchial biopsies (TBB) and microbiologic results from surveillance bronchoscopies. We aimed to define reference values that would allow distinction between four specific groups “ACR”, “infection”, “combined ACR and infection” and “no pathologic process”. No definitive pattern was identified. Given the overlap between groups, these four cytokines are not suitable diagnostic markers for ACR or infection after lung transplantation. Keywords Abbreviations acute cellular rejection antibody-mediated rejection bronchoalveolar lavage chronic lung allograft dysfunction cytomegalovirus chronic obstructive pulmonary disease C-reactive protein enzyme-linked immunosorbent assay interferon gamma interleukin interquartile range International Society for Heart and Lung Transplantation lung transplant recipient transbronchial biopsy tumor necrosis factor alpha"

"Authorship NES conducted data collection and statistical analysis, provided draft versions and revised the manuscripts. EP supervised the laboratory analyses, reviewed all versions of the manuscript and assisted to write the final version. SH performed statistical analysis and assisted to write the final version. CB and MK reviewed all versions of the manuscript and assisted to write the final version. LCH designed the project, revised all versions of the manuscript and assisted to write the final version. CAR designed and supervised the project, revised all versions of the manuscript and provided the final version. All authors read and approved the final manuscript. Funding sources This study was partially funded by Lunge Zürich . Disclosure statement The other authors have no conflicts of interest to disclose. This work was supported by a grant from Lunge Zürich (grant number 2017-01). Credit author statement NES conducted data collection and statistical analysis, provided draft versions and revised the manuscripts. EP supervised the laboratory analyses, reviewed all versions of the manuscript and assisted to write the final version. SH performed statistical analysis and assisted to write the final version. CB and MK reviewed all versions of the manuscript and assisted to write the final version. LCH designed the project, revised all versions of the manuscript and assisted to write the final version. CAR designed and supervised the project, revised all versions of the manuscript and provided the final version. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."

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"NO-CC CODE"

"no"

"2023-06-06 23:35:01"

"Cytokine. 2020 Jan 7; 125:154794"

"oa_package/73/b5/PMC7128992.tar.gz"

"PMC7129780"

"31479875"

"Introduction Interferons (IFNs) are a group of cytokines that serve as the first line of defense against viruses. In addition to their protective role against viral infection, the interferon (IFN) family - consisting of types I, II, and III IFNs, have numerous additional functions that influence cellular growth and immune surveillance against tumor cells [1] , [2] , [3] . All three IFN members activate the JAK/STAT pathway and induce interferon-stimulated gene (ISG) expression by binding to their respective receptors: IFNαR1 and IFNαR2 for type I interferon (IFNα/β), IFNγR1 and IFNγR2 for type II interferon (IFNγ), and IFNλR1 and IL10Rβ for type III interferon (IFNλ1~4) [1] , [4] . In contrast to types I and II, type III IFN was only recently identified and plays not only antiviral functions but also novel immunomodulatory functions in oncology and autoimmune diseases [5] , [6] . IFNλ1~3 were identified through computational based prediction from genome sequencing [7] , [8] and IFNλ4 was discovered in genome-wide association studies (GWAS) on hepatitis C virus (HCV)-infected patients [9] . The ΔG allele of a dinucleotide genetic variant (rs368234815) that is upstream of the IFNL3 locus on chromosome 19 creates the functional IFNλ4, while the TT allele leads to a frameshift, thereby rendering it a pseudogene [9] . Interestingly, HCV patients with the ΔG allele and hence expressing IFNλ4, responded poorly to PEGylated-IFNα-ribavirin treatment as compared to patients with the TT allele [10] . However, IFNλ4 still induces the major hepatic ISG expression during chronic HCV infection and is able to drive the anti-viral response against other viruses such as the MERS-CoV in vitro [11] . Similar to IFNα (Roferon-A for hairy cell leukemia) and IFNβ (Avonex for multiple sclerosis), successful phase 2 clinical trials of PEGylated IFNλ1 against hepatitis D virus (HDV) infection highlight the pharmaceutical potential of the IFNλ family. Previously, the transient expression of wild-type IFNλ4 in mammalian cells failed to produce significant amounts of recombinant IFNλ4. It was suggested that a weak signal peptide in IFNλ4 may be responsible for its impaired secretion and that proper glycosylation of IFNλ4 may be required for its secretion [9] . Although recombinant IFNλ4 can be purified from a bacterial expression system through refolding the inclusion body [11] , a lack of glycosylation may affect the efficacy of IFNλ4. Recently, glyco-engineering, which introduces new glycosylation sites or alters the glycan composition of CHO cells, has been widely used to produce improved therapeutic proteins, because glycan moieties can affect various protein properties, such as solubility, stability, in vivo activity, and serum half-life. For example, improved half-life and productivity are obtained from glyco-engineered hIFNβ-1a and hIFNα [12] , [13] . Moreover, increased secretion of lipase, cutinase, llama V HH antibody, and macrophage inhibitory cytokine 1 results from the addition of a single N-glycosylation site [14] , [15] . Therefore, we propose that glyco-engineering IFNλ4 is a viable option for improving its expression level and possibly altering other properties. In this study, we used mutagenesis to introduce new potential N-glycosylation sites based on the model structures of the IL10Rβ-IFNλ4-IFNλR1 complex. Our results indicate that, among several IFNλ4 variants, three - L28N, P73N, and L28N + P73N - exhibited enhanced productivity, although only P73N was glycosylated de novo . Moreover, these HEK293-expressed IFNλ4 variants retained their binding affinity to the specific IL10Rβ and IFNλR1 receptors, and showed a more potent IFNλ4-mediated signaling and antiviral activity than did E. coli -derived IFNλ4 (eIFNλ4)."

"Materials and methods Modeling process The human IFNλ4 amino acid sequence (22~179, NCBI Accession Number: AFQ38559.1) was used in SWISS-MODEL homology modeling with three templates (PDB code: 5T5W.1.C, 3OG6.1.A, 3OG4.1.A). The model with the highest QMEAN-Z (Qualitative Model Energy ANalysis-Z) score (−2.56) was aligned to the IL10Rβ-IFNλ3-IFNλR1 structure (PDB code: 5T5W) to create the IL10Rβ-IFNλ4-IFNλR1 model. Cell lines, cell culture, and reagents Expi293F (#A14527, Gibco®) cells were cultured according to ATCC guidelines and used within 6 months of receipt. They were maintained in suspension in Expi293F expression medium (#14351, Gibco®) at 37 °C and 8% CO 2 with 125 rpm agitation. Huh-7.5 cells (Apath) were maintained at 37 °C with 5% CO 2 in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (WelGENE), 4.5 g/l glucose, L-glutamine, and 1% penicillin/streptomycin (WelGENE). Small-interfering RNAs (siRNAs) against IFNλR1 and scrambled sequences were obtained from Santa Cruz Biotechnology. Transfection of IFNλR1 siRNA was performed using lipofectamine RNAi MAX (Invitrogen). Recombinant IFN-α-2a was obtained from PBL Assay Science, recombinant IFN-β was obtained from PeproTech, and recombinant human IFNλ1 (1598-IL), λ2 (8417-IL), λ3 (5259-IL), and eIFNλ4 (9165-IL) were obtained from R&D Systems. Expression and purification of recombinant proteins Gene encoding human IFNλ4 (1~179) was cloned into a modified pcDNA3.1 (#V79020, InvitrogenTM) containing a C-terminal 6x-His tag. IFNλ4 variants were generated by site-directed mutagenesis (QuikChange site-Directed Mutagenesis Kit, #200519, Agilent) using the IFNλ4 wild-type construct as the PCR template. The primers for site-directed mutagenesis are listed in Supplementary Table 1 . For IFNλ4-Protein A expression, the C-terminal 6x-His in the IFNλ4 constructs were replaced with a Protein A gene derived from PEZZ18 (#VPT4033, GE Healthcare life Sciences). A thrombin cleavage sequence (LVPRGS) was introduced between the IFNλ4 genes and the Protein A gene using the PCR primer, in order to remove Protein A. IFNλ4 wild-type and variants containing 6x-His or Protein A were transfected into Expi293F cells using ExpiFectamine 293 Transfection Kits (#A14524, Invitrogen M ), following the manufacturer’s protocol. For the purification of IFNλ4 variants, the supernatant containing secreted IFNλ4-Protein A was loaded onto IgG Sepharose resin (#17096902, GE Healthcare Life Sciences). After three washes with 1x PBS, the protein-bound resins were incubated overnight with thrombin (1% (v/v) in 1x PBS) at 4 °C to remove the C-terminal Protein A tag. Eluted IFNλ4 variants were subsequently purified by gel-filtration chromatography in a Superdex 200 Increase 10/300 GL column (#28990944, GE Healthcare Life Sciences) equilibrated with 1x PBS. Immunoblotting The cells were lysed with RIPA buffer (Thermo Fisher Scientific) to prepare total cell lysates. Ten micrograms of each cell lysate were loaded on SDS-PAGE gels prior to immunoblotting. The antibodies used for immunoblotting were: IFNλ4 (1:200, mouse, Millipore MABF227), IFNλ4 (1:200, rabbit, Abcam ab196984), STAT1 (1:1000, rabbit, BD Biosciences 610120), PY-STAT1 (1:1000, mouse, BD Biosciences 612233), STAT2 (1:1000, rabbit, Santa Cruz Biotechnology sc-476), IRF9 (1:1000, rabbit, Santa Cruz sc-496), SOCS1 (Abcam #62584), USP18 (Cell Signaling Technology #4813), horseradish peroxidase (HRP)-conjugated rabbit IgG (1:5000, Abcam ab97051), and HRP-conjugated mouse IgG (1:5000, Abcam ab97023). PNGase F treatment N-glycans of IFNλ4 were removed using a PNGase F kit (#P0704S, New England Biolabs) according to the manufacturer’s instructions. Briefly, IFNλ4 variants were boiled with Glycoprotein Denaturing Buffer (10×) and chilled on ice. GlycoBuffer(10×), NP-40(10×), and 1 μl of PNGase F were added onto denatured proteins and the mixture was incubated at 37 °C for 1 h before the Western blot analysis. Glycosylation site analysis The glycopeptides resulting from non-specific digestion were prepared as previously described [16] . Briefly, 50 μg/μL IFNλ4 variants were incubated with 50 μg/μL pronase E for 1 h at 37 °C. The digested glycopeptides were enriched by graphitized carbon solid-phase extraction (PGC-SPE) and analyzed by nanoLC-Chip Q-TOF MS (Agilent Technologies). The LC-MS and MS/MS data were processed and interpreted as previously described, using MassHunter Qualitative Analysis software (version B.07.00, Agilent Technologies) and GP Finder software [17] . Determination of binding kinetics The IFNλ4 variant binding kinetics to IFNλR1 and IL10Rβ were measured by biolayer light interferometry on a BLItz system (ForteBio, Pall Life Sciences). The mixtures were agitated at 2200 rpm in washing buffer (200 mM NaCl, 20 mM Tris-HCl pH 8, 5% glycerol, 0.01% Tween-20). Assays were performed at room temperature. Biotinylated IFNλ4, at concentrations of 0.25 mg/ml, were loaded onto the surfaces of streptavidin biosensors (ForteBio) for 1 min, followed by washing of the loaded biosensors for 2 min with washing buffer (200 mM NaCl, 20 mM Tris-HCl pH 8, 5% glycerol, 0.01% Tween-20) to remove any unbound protein. The biosensor tips were immersed in drops containing indicated concentration of IFNλR1 and IL10Rβ (500, 1000 and 2000 nM). Associations (on rate, k on ) were measured over a 2 min interval. The sensors were subsequently immersed in washing buffer for 2 min to measure dissociation (off-rate, k off ). K D , measured in nanomoles, was calculated as the ratio of off-rate to on-rate. The resulting data were analyzed by fitting to a 1:1 ligand model with the global fitting function. Production and infection of cell culture-derived HCV (HCVcc) The Japanese fulminant hepatits-1 (JFH-1) strain (genotype 2a) of HCVcc was produced as described previously [18] . DMEM containing 5% human serum was used to culture the Huh-7.5 cells, in order to produce highly infectious JFH1 HCVcc. HCVcc infectivity was quantified by a colorimetric focus-forming assay, as described previously [19] . Huh-7.5 cells were infected with JFH-1 HCVcc at 0.5 multiplicity of infection (MOI). RNA extraction and real-time quantitative PCR Total RNA isolation and TaqMan real-time quantitative PCR were performed as described previously [20] . In brief, total RNA was isolated with GeneAll RibospinTM (GeneAll), after which TaqMan Gene Expression Assays (Applied Biosystems) were used to determine the mRNA levels of the target genes. Quantification of intracellular HCV RNA copies was performed as described previously [20] . The results were standardized to the mRNA levels of GAPDH and the data are presented as means ± standard error of the mean. TaqMan Assay (Applied Biosystems) used in this study are: IFNLR1 (Hs00417120_m1), ISG15 (Hs01921425_s1), MX1 (Hs00895608_m1), SOCS1 (Hs00705164_s1), USP18 (Hs00276441_m1), GAPDH (Hs02758991_g1). IFNL proteins (R&D Systems) used in this study are: IFNL1 (1598-IL), IFNL2 (8417-IL), IFNL3 (5259-IL), eIFNL4 (9165-IL). Statistical analysis Data from experiments with cell lines are presented as means ± standard error of the mean. Unpaired t -tests or two-tailed Mann-Whitney U-tests were performed for statistical analysis. All of the analyses for real-time quantitative PCR were performed with GraphPad Prism version 7.01. P values less than 0.05 were considered to be statistically significant."

"Results Design and expression of IFNλ4 variants The low affinity of wild-type IFNλ to its receptor, IL10Rβ, hampers the production of the stable ternary complex - IL10Rβ-IFNλ-IFNλR1. Therefore, only the structures of IFNλ3 alone [21] or IFNλ1 in complex with IFNλR1 [22] have been determined. Recently, Mendoza, et al ., introduced affinity-enhancing mutations on IFNλ3 which stabilized its interaction with IL10Rβ, and elucidated the crystal structure of the type III interferon signaling complex, IL10Rβ-IFNλ3-IFNλR1 (PDB code: 5T5W) ( Fig. 1 A) [23] . Although IFNλ4 shares only ~30% sequence identity with IFNλ1~3, the sequence alignment of IFNλ1~4 suggests that IFNλ4 interacts with IFNλR1 and IL10Rβ in a similar manner as the IL10Rβ-IFNλ3-IFNλR1 ternary complex [23] for two reasons. First, the amino acids of the IFNλ family that are critical for IFNλR1 binding are well-conserved in IFNλ4 (P37, L40, K44, R47, D48, I108, F159, and R163) ( Fig. 1 B). Second, hydroxyl groups of several aromatic residues of IL10Rβ (Y59, Y82, Y140, and W143) form a hydrogen bonding network with IFNλ3 (S44, L45, Q48R, and E106D); these are also well conserved in IFNλ4 (S34, L35, R48, and Q100). Therefore, we modeled the IFNλ4 structure using the crystal structure of IFNλ3 and IFNλ1 ( Fig. 1 A) and structurally aligned it to the IL10Rβ-IFNλ3-IFNλR1 structure to build the IL10Rβ-IFNλ4-IFNλR1 model ( Fig. 1 B). Interestingly, the model structure of IL10Rβ-IFNλ4-IFNλR1 indicates that critical hydrophobic pockets for harboring the hydrophobic residues of IL10Rβ (Y82 and W143) are well maintained on the surface of IFNλ4 ( Fig. 1 C). Using the IL10Rβ-IFNλ4-IFNλR1 model structure, we searched the new N-glycosylation candidate sites of IFNλ4 based on three criteria. First, the sites had to be outside the receptor binding region to minimize the change in the receptor-ligand binding and signal activation. Second, they had to be exposed to the solvent to allow access to oligosaccharyltransferase (OST), which catalyzes the initial transfer of glycan from the lipid-linked oligosaccharide onto the substrate asparagine [24] , [25] . Third, the consensus sequence (NXS/T, X = any amino acid except proline) had to be achieved by single point mutation to minimize the structural distortion caused by the mutation. Only six candidate sites were available that met all three criteria: L28N, A54N, P73N, H97N, K154N, and A173N ( Fig. 1 A and B). We named them M1 ~ M6, respectively. Next, we examined the expression levels of each of the IFNλ4 variants (M1 ~ M6) by western blot and found that two IFNλ4 variants, M1 (L28N mutation) and M3 (P73N mutation), resulted in enhanced protein expression ( Fig. 2 A). Interestingly, only M3 showed the prominent up-shift in SDS-PAGE that indicates successful hyperglycosylation. We also checked the expression level of the double mutants (L28N and P73N, M7), showing further enhanced protein expression compared to M1 and M3 variants ( Fig. 2 A). The constructs used in the western blot for hit discovery carried a C-terminus 6x Histidine tag, which may interfere with proper secretion of the protein, given the extensive distribution of positively-charged amino acids in IFNλ4. Therefore, we substituted the 6x histidine tag with a protein A tag and purified three IFNλ4 variants (M1, M3, and M7) using affinity chromatography followed by thrombin digestion, in order to remove the protein A tag and subsequent size exclusion chromatography. Final IFNλ4 variants (M1, M3, and M7) were analyzed by SDS-PAGE and Coomassie blue staining under reducing and non-reducing condition. The resulting bands indicate that three IFNλ4 variants (M1, M3, and M7) are monomer ( Fig. 2 B). The elution profile of standard proteins indicates that each monodispersed peak corresponds to the IFNλ4 variants (~44 kDa) ( Fig. 2 C). Most likely, this oversized elution is due to the presence of N-glycosylation on IFNλ4 variants, which was confirmed by the results shown in the following section. Identification of N-glycans on IFNλ4 variants To identify the presence of N-glycans on the three IFNλ4 variants, we treated them with PNGase F and compared their sizes with SDS-PAGE. The M3 (P73N) and M7 (L28N + P73N) IFNλ4 variants were located at higher molecular weight positions compared to the M1 (L28N) IFNλ4 variant. After de-glycosylation with PNGase F, however, the molecular weight of the three IFNλ4 variants decreased to the same level, indicating the presence of N-glycans in all IFNλ4 variants, but the status or position of M1 N-glycosylation may be slightly different from those of M3 and M7 IFNλ4 variants ( Fig. 3 A). We used mass spectrometry to determine the exact position of N-glycans on IFNλ4 variants [26] . Briefly, purified IFNλ4 variants were treated with pronase E to produce glycopeptides, and ultimately to determine the glycosylation site. The glycopeptides were then separated and analyzed by nanoLC-Chip Q-TOF MS. The LC/MS data indicate that the mutated L28N in M1 and M7 IFNλ4 variants were not glycosylated, whereas an original N-glycosylation site, Asn61, and mutated P73N were fully occupied by N-glycans ( Fig. 3 B–D). These are in accordance with the PNGase F treatment results, where M1 (L28N) migrated more quickly than either M3 (P73N) or M7 (L28N + P73N). Receptor binding affinity and biological activity of IFNλ4 variants To investigate whether the mutation and the additional glycan on IFNλ4 variants affect their binding to their receptors, IL10Rβ and IFNλR1, we examined the in vitro binding affinity of IFNλ4 variants to IL10Rβ and IFNλR1 by Biolayer Light Interferometry (BLI) and then compared them with that of IFNλ4 WT purified from E. coli (eIFNλ4). Similar to eIFNλ4, the three IFNλ4 variants properly bound to their receptors and their binding affinities to IFNλR1 were higher than to IL10Rβ ( Fig. 4 ). Moreover, our variants had a slightly higher affinity towards IL10Rβ than the eIFNλ4 does (For IL10Rβ, K D M1 = 49 nM, K D M3 = 51 nM, K D M7 = 49 nM, K D eIFNλ4 = 71 nM), while their binding affinity to IFNλR1 was similar to each other (For IFNλR1, K D M1 = 14 nM, K D M3 = 22 nM, K D M7 = 17 nM, K D eIFNλ4 = 19 nM). The modifications elicited by mutation and glycosylation does not inhibit their interaction with their specific receptors, even further stabilize the interaction between IFNλ4 and IL10Rβ. In order to determine whether the mutation and additional glycan on the IFNλ4 variants affected their functional activity, we investigated their IFNλR1-dependent phospho-STAT1 signaling upon treatment with the IFNλ4 variants. Our results indicate that treatment with the M1, M3, and M7 IFNλ4 variants also induced phosphorylation of STAT1, just as in other type III interferons, IFNλ1~3, and that the suppression of IFNλR1 expression by small interference RNA specific to the IFNλR1 gene (siIFNλR1) abolished the phosphorylation of STAT1, even after treatment with the IFNλ4 variants ( Fig. 5 A). IFNλ4 stimulation reportedly leads to the assembly of the ISGF3 transcription factor complex, which consists of phospho-STAT1, phospho-STAT2, and IRF9 and induces the expression of ISG15 [27] , which is critical for anti-viral activity [28] . We showed that the M1, M3, and M7 IFNλ4 variants also induced the expression of ISG15 and inhibited HCV replication in HCV-infected Huh-7.5 cells ( Fig. 5 B and 5C). Interestingly, the M1, M3, and M7 IFNλ4 variants showed a significantly more potent ISG induction and anti-viral activity than eIFNλ4. Prolonged exposure to IFNλ proteins induces the production of unphosphorylated ISGF3 (U-ISGF3) consisting of STAT1, STAT2 and IRF9 without tyrosine phosphorylation while the expression of phosphorylated ISGF3 are diminished [18] . As a result, the upregulation of the U-ISGF3-specific set of genes, such as Mx1, is maintained long-term. In order to assess whether the M1, M3, and M7 variants display a similar functionality during prolonged treatment, we evaluated the protein levels of the U-ISGF3 components. The protein levels of STAT1, STAT2, and IRF9 were equally upregulated by all IFNλ4s ( Fig. 5 D). Nevertheless, our IFNλ4 variants maintained the upregulation of Mx1 more robustly than did eIFNλ4, but IFNλ1, 2 and 3 maintained more strongly the upregulation of Mx1 expression compared to our IFNλ4 variants ( Fig. 5 E). Previously, eIFNλ4 is shown to induce the expression of negative regulators of IFN signaling [27] , [29] such as SOCS1 and USP18. To assess the effect of glycosylation on this functionality, we examined the expression level of SOCS1 and USP18 upon the treatment of M1, M3 and M7 variants on Huh7 cell line. USP18 was significantly increased upon treatment of IFNλ1, 2 and 3. While eIFNλ4 resulted in slight increase of USP18, our IFNλ4 variants (M1, M3 and M7) showed a comparable activity to IFNλ1, 2 and 3 ( Fig. 5 F). The protein expression of SOCS1 was not significantly increased by the treatment of any form of IFNλs ( Fig. 5 F). However, the mRNA expression of SOCS1 was slightly upregulated by IFNλs, although there was no significant difference among IFNλs ( Fig. 5 G). These results suggest that our structure-based approach on selecting de novo glycosylation maintained the biological activity of IFNλ4 and our IFNλ4 variants expressed from HEK293 have superior activity compared to eIFNλ4."

"Discussion Until recently, research on IFNλ4 and its use as a clinical therapeutic was hindered by the inability to obtain appreciable amounts of this protein. Purification of bacterial-derived recombinant IFNλ4 through refolding [11] still poses a number of problems, including the complexity of purification steps, lack of glycosylation, and endotoxin contamination. Our study is an example of how glycoengineering aided by structural information can be used to overcome such limitations; it is the first to report the successful production of IFNλ4 protein from a mammalian cell line with enhanced properties compared to eIFNλ4 and requiring a simpler purification protocol. There are several possibilities as to why our IFNλ4 variants displayed enhanced expression and potency. First, the presence of acidic N-glycans may stabilize the protein through a charge-balance, since IFNλ4 is unusually abundant with positively charged amino acids (~23%). Second, considering how the immune response is more actively triggered by our variants without critically affecting the receptor binding activity, compared to eIFNλ4, extra N-glycans may have extended the half-life, as in other reports [12] , [30] , thereby increasing the fraction of functional protein during treatment. Finally, since N-glycosylation in eukaryotes is co-translational [31] , the protein folding may have also been affected. Nevertheless, the mechanism behind the enhanced expression induced by unmodified L28N remains unanswered. We speculate that L28 may serve as the hydrophobic aggregation nuclei interacting with nearby hydrophobic residues, such as L29 or Y32 [32] , [33] ; perhaps L28N alleviates this effect. This study is unique, in that we were able to successfully identify a viable de novo N-glycosylation site by structural elucidation of glyco-peptides. We used endogenous serine, threonine, or asparagine to introduce the glycosylation modification, which minimized the mutation-induced structural distortion. Other IFNλ4 candidates containing N-glycosylation sites at random locations were also tested (data not shown) but no additional modification or expression changes were observed, indicating the efficiency of our structure-oriented approach. However, glyco-peptides containing L28N were not detected by mass spectrometry, suggesting that the site may have remained unmodified. According to the recent cryo-EM structure of oligosaccharyltransferase (OST) [34] , [35] , [36] , which catalyzes the initial transfer of glycan from the lipid-linked oligosaccharide onto the substrate asparagine, substrate binding to the catalytic subunit, STT3, requires structural flexibility near the glycosylation sequence of the substrate. Our IFNλ4 model suggests that P73 is located on the flexible loop, while other eliminated candidate sites (A173 and K154) are part of the α-helix. This may partly explain the successful glycosylation of P73N. On the contrary, L28N may not be physically accessible by OST. Elucidating the structure of IFNλ4 may provide insights to this hypothesis. A number of interferons are already targets of drug development, due to their ability to generate strong antiviral and antitumor responses or to modulate immune responses. IFNα and its PEGylated variants are used against cancers such as hairy cell leukemia (Roferon A), melanoma (Multiferon), and AIDS-related Kaposi's sarcoma (Intron A) [37] , [38] , [39] . IFNβ is a well-known treatment for multiple sclerosis, with a number of different versions available (Rebif – IFNβ 1a, liquid form, Avonex – IFNβ 1a, lyophilized, Cinnovex – IFNβ 1a, biogeneric, Betaseron – IFNβ 1b, Plegridy – PEGylated IFNβ 1a) [40] , [41] , [42] , [43] . Similar to this, IFNλs also have a therapeutic potential, because it has been shown that IFNλs can protect hosts from various viruses, including influenza virus, West Nile virus, norovirus and rotavirus [38] , [39] , [40] , [41] . It will be very interesting to test if glycosylated IFNλs exert anti-viral activity in hosts infected by such viruses. In this regard, a recent study demonstrated that IFNλs can suppress influenza virus without the inflammatory side effects of IFNα [44] . However, it was previously shown that the expression of functional IFNλ4 is associated with unresponsiveness to IFNα treatment among HCV-infected patients [9] , and a subsequent study showed that long-term exposure to IFNλ4 leads to cellular unresponsiveness to IFNα treatment by upregulation of USP18 or SOCS1 [27] , [29] , indicating that IFNλ4 treatment may be detrimental to virus-infected patients. On the other hand, our result shown in Fig. 5 C indicates that IFNλ4 treatment can directly suppress HCV replication. Whether IFNλ4 treatment will be beneficial or detrimental to virus-infected patients might be determined by duration of the treatment and use of IFNα following IFNλ4 treatment. Although much further research and insight into the mode of action of IFNλ4 is required to understand if its effects are beneficial or detrimental to human health, our engineered IFNλ4 variants can be utilized as an alternative platform of IFNλ4 wild-type."

""

"These authors contributed equally to this work. Graphical abstract Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression in vitro . Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced de novo N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptor-mediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4. Keywords"

"Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."

"Supplementary material The following are the Supplementary data to this article: Acknowledgments This work was supported by the 10.13039/501100003725 National Research Foundation of Korea (NRF) grant (2015M3A9B5053960 to H.M.K. and H.J.A) and the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI18C0135 to H.M.K.). Author contributions J.H.C., S.H.H., H.J.A, E.C.S., and H.M.K. designed the experiments; J.H.C., S.H.H., and N.S. contributed to the experimental work; J.H.C., P.L., E.C.S., and H.M.K. wrote and edited the manuscript. E.C.S. and H.M.K. supervised the project. All authors reviewed the manuscript."

"NO-CC CODE"

"no"

"2023-06-06 23:35:02"

"Cytokine. 2020 Jan 31; 125:154833"

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"PMC7132398"

"25175674"

"Introduction Three quarters of all emerging infectious diseases are zoonoses; diseases that are transmissible between vertebrate animals and humans ( Taylor et al., 2001 ). The management of such diseases usually benefits from a One Health multidisciplinary approach as it requires the collaboration of veterinary and medical professionals to mitigate biosecurity and public health risks. Many zoonoses have been known for decades or even centuries (e.g., Q fever, brucellosis, leptospirosis, salmonellosis) while others have only been recently recognised (emerging zoonotic diseases). Some emerging zoonoses spill over into humans and progress no further. Others spill over and succeed in making the next transition into human-to-human dissemination, becoming a major threat to human health (e.g., Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome (SARS), swine influenza H1N12009, Ebola virus). Although some of the emerging zoonoses that remain confined to the first generation of human victims have a low-incidence, they remain a public health threat because of the severity of their pathogenicity in humans ( Belay and Monroe, 2014 ). Hendra virus (HeV) is a good example of such a zoonosis. It emerged in humans in Australia in 1993 after spilling over from flying foxes ( Pteropus spp.) to horses to humans ( Murray et al., 1995a , Murray et al., 1995b , Halpin et al., 2000 , Halpin et al., 2011 ). Since its emergence it has remained a uniquely Australian zoonosis where 49 equine outbreaks have occurred on the eastern coast between Far North Queensland (QLD) and Northern New South Wales ( Department of Agriculture, Fisheries and Forestry Queensland (DAFF QLD), 2014 ). During this time, 91 horses have been suspected of HeV infection, 71 of which were confirmed positive; while only seven people have been infected ( Murray et al., 1995a , Murray et al., 1995b , O'Sullivan et al., 1997 , Hanna et al., 2006 , Field et al., 2010 , Playford et al., 2010 , Mahalingam et al., 2012 , DAFF QLD, 2014 ). Compared to pandemic Influenza and SARS, HeV could be considered a minor threat to public health. However, HeV has a 57.1% case fatality rate in humans and all four who died of HeV had professional or direct caring roles for the ill horses. Two were equine veterinarians, one was a person assisting a veterinarian during a horse necropsy, and one was a horse trainer. Another veterinarian, a veterinary nurse, and a stable hand also became infected with HeV but survived. Those who became infected with HeV, did so through close exposure to infectious blood and/or other bodily fluids such as respiratory secretions from an infected horse ( Animal Health Australia, 2013 , DAFF QLD, 2014 ). Currently there is no cure available for those who become infected with HeV. A human monoclonal antibody has been shown to neutralise HeV in primates and has been used as experimental prophylaxis in humans but it is not currently licenced or available commercially ( Bossart et al., 2011 ). Consequently public health, biosecurity and occupational health and safety government authorities primarily targeted their HeV preventative recommendations to equine veterinarians and their staff. In 2010, all private veterinarians registered with the Veterinary Surgeons Board of Queensland (VSBQ) received a comprehensive information package about HeV and its management ( DAFF QLD, 2010 ). Up until 2011, the prevention of HeV was solely based on avoiding exposure, through contact with horses potentially infected with the virus, by implementing adequate infection control (IC) measures such as following hygiene and quarantine protocols and the use of personal protective equipment (PPE). In October 2012 a new HeV vaccine for horses was also released and promoted as a One Health measure that would protect both horses and the people coming into contact with horses, and in particular horse owners and veterinary personnel ( Pallister et al., 2011a , Pallister et al., 2011b , Animal Health Australia, 2013 , Middleton et al., 2014 ). However, after more than a year in circulation the vaccine uptake seems to have been moderate despite the initial motivation of veterinarians to encourage horse owners to vaccinate their horses ( Mendez et al., 2013a , DPI NSW, 2013 . Veterinary IC therefore remains a major component of the management of HeV in private practices providing equine veterinary services. In the winter of 2011, as part of a cross-sectional study of private veterinarians registered in QLD we investigated: HeV management strategies implemented by eligible veterinarians including their use of PPE, with the aim of identifying the strategies implemented by private veterinarians who treated horses; and to determine if they modified their use of PPE depending on the health status of their equine patients."

"Methods Study design This study was conducted as a cross-sectional study of private veterinarians providing equine services in QLD, Australia. Eligibility criteria for participation were as follow: (1) being a qualified veterinarian; (2) being registered in QLD; (3) working in private practice in QLD; and (4) to have provided veterinary services to at least one horse in the previous 12 months. All private veterinarians registered with the Veterinary Surgeons Board of Queensland (VSBQ) and working in private veterinary practices in QLD were invited to participate and self-select as providers of veterinary services to horses. Participation was voluntary and participants could withdraw from the study at any time. This study was conducted with the approval of the James Cook University Human Ethics Committee (Ethics Approval No. H3687) Questionnaire design The questionnaire used for this study was based on the results from a previous qualitative study which explored the HeV-risk related perceptions and barriers to IC and HeV management in equine veterinary practices in QLD between 2009 and 2010 ( Mendez et al., 2012a , Mendez et al., 2012b , Mendez et al., 2013b ). The questionnaire also took into account the HeV management recommendations and Workplace Health and Safety (WHS) regulations in place at the time of the study design ( DAFF QLD 2010 ; Workplace Health and Safety Queensland (WHS QLD); 2011a ). The questionnaire was piloted with 6 eligible veterinarians within the target population prior to its implementation. The questionnaire comprised of a number of socio-demographic, professional and practice profile questions and a number of multiple choice and open-ended questions on the topics of HeV management: management plan; IC facilities in the veterinary practice; HeV field kit; and HeV risk communication with clients. Participants’ geographic locations within QLD were categorised according to their postcodes into two regions: Brisbane and Moreton, and other; and five Accessibility/Remoteness Index of Australia (ARIA) categories: highly accessible, accessible, moderately accessible, remote, and very remote. ARIA categories are based on the road distance from the closest service centres ( Australian Institute of Health and Welfare, 2004 ). Moreover, participants were asked how often they used PPE depending on the health status of a horse: a healthy horse (HH); a sick horse (SH); or when they were conducting a necropsy on a dead horse (NH). Participants were also asked to detail which items of PPE they used in each of the different horse health scenarios as well as in the case a horse was suspected to be infected with HeV (HeVH). Participants could choose PPE items from 9 different types of PPE: body (overalls, gown or apron), head (hat), feet (gumboots or boot covers), oro-nasal only (surgical mask, surgical mask with fluid barrier or particulate mask [N95/P2]), oro-nasal and ocular (surgical mask with eye shield or face shield), ocular only (goggles), hand (gloves or arm length gloves) PPE; dressing to cover open wounds or skin abrasions; and powered air respirator (PAR). For each scenario respondents scored one point for each type of PPE they used. Questionnaire implementation The questionnaire was first mailed out in June 2011 to 1604 veterinarians registered with the VSBQ and who were identified as working in private practice in QLD. Three reminders were subsequently mailed out in July, August and September 2011. Each mail out contained an information page, the questionnaire and a pre-paid envelop to return participants’ responses. All responses were de-identified upon reception. Data management and analysis Each questionnaire was attributed a unique identification number and responses were collated in Excel (Microsoft. Released 2010) before being transferred into SPSS (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.) for analysis. Categorical data were reported using percentages. Numerical data were reported using mean and standard deviations (SD) when symmetrical, and median and interquartile range (IQR) when skewed. Participants who “always or sometimes” used PPE in a range of scenarios related to the health status of horses were compared to those who used PPE “less often” using Pearson's Chi-square, Chi-square for trend, and, Fisher's exact test. Participants’ PPE usage score according to the health status of horses were compared using a Friedman's test. Participants with a PPE usage score above or equal to the median score in a range of scenarios related to the health status of horses were also compared to those who scored below the median score using Pearson's Chi-square. Bivariate analyses were conducted with respect to socio-demographic, practice, professional and HeV experience characteristics. The assumptions for these statistical tests were met. An alpha level of 0.05 was used for all statistical analyses."

"Results The socio-demographic, professional and HeV experience characteristics of 204 respondents have been presented elsewhere ( Mendez et al., 2013a ). However 4 participants, who answered the questions about HeV vaccination for horses in Mendez et al. (2013a) , were excluded from the present data analysis as they did not answer the questions about HeV management or were in fact ineligible because they had retired from private practice. Additionally, not all participants answered all questions. Socio-demographic and veterinary education ( Table 1 ) Of the 200 respondents, 96 were male (48%); 116 were 40 years or younger (58%); 156 (78%) graduated from a QLD University. Overall, 70 (34.3%) worked in the Brisbane and Moreton region and 87 (43.5%) were from highly accessible to accessible ARIA ( Australian Institute of Health and Welfare, 2004 ). Professional, veterinary practice and HeV management experience ( Table 2 ) The majority of the respondents worked full-time (89.5%) in mixed practice (79.3%) and provided equine services at least once a week (77.4%) to mostly hobby farms (82.4%) and farms (77.9%) and pony clubs (51.8%). Sixty-six percent had experience dealing with horses potentially infected with HeV. However, only 38.5% had attended an IC/HeV management training session in the previous 12 months. The procedures mostly performed on horses involved: wound management (97.9%), the oro-nasal area (71.6%) and uro-genital area (57.7%). Less than a fifth of respondents carried out necropsies on horses (17.5%). Of the 124 respondents who answered the questions relating to IC control facilities and equipment available at their practice, most reported to have access to sharp disposal units (95.2%) and more than half had access to an autoclave for equipment sterilisation (67.7%). More than half of those who did not answer these questions did not give any further explanation (45/76), while the others (31/76) explained that they either only saw horses in the field or that their practice was purely ambulatory and therefore did not have any clinical premises. However, all the other IC facilities and equipment were much less available in the practices where respondents worked. For example, only 58.9% of participants reported to have a dedicated hand washing station in the practice; 50.8% had access to a biological waste disposal unit and only 34.7% had a quarantine/isolation stable. HeV management strategies implemented in the winter of 2011 ( Table 3 ) The majority of participants worked in a practice that had a HeV management plan (83.1%) but only 58.1% had an in-house HeV specific set of policies/standards procedures. However, in 71.2% (136/191) of cases the HeV management plan included a set of official guidelines (mostly those provided by the QLD government (80.7%; n = 88)) and a dedicated HeV field kit (79.1%; n = 191) (mostly put together in house (63.7%) rather than a commercial kit (30.6%)). Many of the other elements recommended by government authorities were not consistently included in the participants’ HeV management plans: 46.6% had a HeV risk assessment plan; 28.4% kept records of HeV training attended by staff; only 22.5% had a HeV case reporting system even though HeV is a notifiable zoonosis ( Animal Health Australia, 2013 , DAFF QLD, 2014 ); 31.9% included information materials about flying foxes for horse owners; and 58.6% included HeV information materials for horse owners. Most HeV field kits contained the following items: PPE for the body (98.3%), the hands (93.9%), the eyes (79.9%) and the feet (74.9%); disinfecting agents (80.4%); sampling kits (77.7%); and waste disposal equipment (76.5%). Field kits were checked for maintenance if not used in only 50.8%. PPE usage according to the health status of horses ( Table 4 ) The PPE usage score of participants significantly increased when the health status of the horse decreased ( n = 169; Friedman test; p < 0.001; not included in Table 4 ). Less than half of the participants (32.4%) always or sometimes used PPE with a healthy horse (HH); while 84.8% did so with a sick horse (SH) and 85.6% used PPE when conducting a necropsy on a horse (NH). The type of PPE used by participants also varied according to the health status of the horse including when the horse was suspected of HeV (HeVH). The median scores for PPE usage were: 2 (IQR = 2; range 0–7/9) with a HH; 4 (IQR = 3; range 0–8/9) with a SH; 6 (IQR = 3; range 0–9/9) when conducting a necropsy on a horse; and 6 (IQR = 2; range 3–9/9) with a horse suspected of HeV. When dealing with a HeVH, participants mostly used: hand (98.9%), oro-nasal (97.2%), body (96.6%), feet (85.5%) and ocular (70.4%) PPE and only 3.9% used PAR; however these PPE items were much less used with a SH not suspected of HeV (80.6%, 59.7%, 67.9%, 46.9%, 35.7% and 0.5% respectively). ARIA categories; IC/HeV management plan, training and experience stratified by usage of PPE according to the health status of the horse ( Table 5 ) Respondents’ usage of PPE did not differ significantly across demographic, professional and practice profiles (data not shown) except for the ARIA regions, IC/HeV management training, access to an in house HeV management plan and having experience with a suspected case of HeV. Of the 64 participants who always or sometimes used PPE when examining a healthy horse, 36 (56.2%) had their practice in a highly accessible or accessible ARIA region compared to 51 of the 134 (38.1%) participants who used PPE less often with a HH ( p = 0.003). Participants who always or sometimes used PPE with a HH (64/200), a SH (168/198) or a NH (176/184) were more likely to have attended an IC and or HeV training programme in the previous 12 months (53.1%; 42.9%; 39.2% respectively) than those who used PPE less often in these scenarios (32.1%; 16.7%; 0% respectively) ( p = 0.005; p = 0.007; p = 0.026 respectively). Similar results were observed with participants who worked in a practice which had a dedicated HeV management plan and those who had experience dealing with potential cases of HeV. Gender; education; IC/HeV management training and experience stratified by score PPE usage according to the health status of the horse ( Table 6 ) Participants’ scores of PPE usage according to the horse's health status did not vary significantly with most demographic, professional and practice characteristics (data not shown). However, when examining a HH those who scored above the median score of PPE usage (61/199) were more likely to be male (59%) than those who scored equally or below the median score (43.5%) ( p = 0.043). When examining a SH, those who scored above the median score for PPE usage (79/196) were more likely to have graduated from a QLD university (86.1%); to have attended IC/HeV management training in the previous 12 months (50.6%); and to have had experience with potentially HeV infected horses (75.6%) than those who scored equally or below the median score (74.4%; 30.8%; 60% respectively) ( p = 0.048; p = 0.005; p = 0.0.24 respectively). "

"Discussion Overall, the results of this study show that by 2011 participating veterinarians worked, for the most part, in veterinary practices that had adopted IC/HeV management strategies and attitudes in line with government recommendations. The frequency of PPE usage; and the number and types of PPE items used varied with the health status of equine patients: the sicker the horses the more PPE items were used, and more often. However, these trends were not consistently observed across all practices and participating veterinarians. Some participants were still using very little PPE when attending sick horses presumably because HeV was not suspected. This may indicate that the initial risk perception when attending a horse plays an important role in determining risk mitigation behaviours; in this case the use of sufficient appropriate PPE. Participants’ behaviours and attitudes towards the use of PPE was also influenced by other factors: gender; university of graduation; access to a HeV management plan in the practice; attendance at a IC/HeV management plan training session in the previous 12 months; and previous experience dealing with suspected cases of equine HeV. HeV management plans Although a majority of participants worked in practices that had a formal HeV management plan, many only included a set of official guidelines and a HeV field kit. This may be less a reflection on the level of commitment of equine veterinarians to HeV mitigation than a reflection of the overall IC and zoonoses management strategies in place in these practices. It may also be an indication that IC for the management of HeV is mostly understood by veterinarians as being the usage of PPE. Similar observations were made during the 2009–2010 qualitative study amongst QLD veterinarians. Infection control in veterinary practices across Australia has been shown to be less than optimal even in specialised equine practices where the risk of exposure to HeV is highest ( Leggat et al., 2009 , WHS QLD, 2011b , Dowd et al., 2013 ). The present study showed a lack of IC related equipment in veterinary practices providing services to equine patients. However, some participants may have misinterpreted questions on this topic as most worked in mixed practices which may only have had clinical premises for attending to small domestic animals while large animals may have been mostly seen in the field. Furthermore, other HeV management plan items such as “staff HeV training record keeping system” and “HeV related occupational health and safety reporting system” may not have been regarded as essential to the practical management of a suspected HeV case as they related to WHS proof of compliance. These items would only be required if a human under WHS veterinary responsibility became exposed to HeV. Compiling and keeping WHS proof of compliance has previously been reported to be burdensome for private veterinary practices to the point that in some cases equine practice was abandoned because the level of liability became too high ( Mendez et al., 2012a ). The most used set of official guidelines for the management of HeV was the one made available by Biosecurity QLD ( DAFF QLD, 2010 ). In 2010, all veterinarians registered and practising in QLD received a comprehensive HeV information and recommendations package from Biosecurity QLD ( DAFF QLD, 2010 ); a move well received by the veterinary profession according to a study conducted by the authors in 2009–2010. Results from both these studies confirm that the government's information campaign of private veterinarians reached a large proportion of its target population. However, our study did not evaluate the effectiveness of this campaign on private veterinarians’ attitudes and behaviours in regard to HeV management. HeV field kits Most practices had HeV field kits put together in house. Commercial kits may have been harder to source, more expensive or deemed incomplete. Field kits were not regularly maintained unless used, possibly because they were not being used frequently, or because many of the items included in the field kits were in fact of a disposable nature. Most kits included most items except PPE for head protection; dressings for wound and skin abrasions; and PAR. It is worth noting that the use of PAR, highly recommended by WHS authorities as the best way to avoid exposure to HeV, is still very low in private practice when dealing with horses, including those suspected of HeV. The access to a seemingly complete HeV field kit, however, cannot be equated to the systematic use of the full range of PPE by veterinarians in the field. Use of PPE Not all participating veterinarians reported using the full panoply of recommended PPE systematically with all horses. The unpracticality of PPE in the field was also found to be an issue common to the management of other non-zoonotic infectious diseases such as equine influenza ( Schemann et al., 2014 ). Furthermore, any additional PPE-related costs had to be justifiable to clients as it affected consultation fees. In order to curb the cost barrier, DAFF QLD introduced a PPE rebate programme in July 2012 ( DAFF QLD, 2012 ). However, the effectiveness of this strategy has not been evaluated. In 2011, when a horse was healthy, male participants were more likely to use more PPE than females. Male veterinarians may have been attending to horses more regularly or may have been carrying out more risky procedures than female veterinarians which would explain their different approach to PPE usage in this case. However, this difference in PPE usage by gender was not carried over in the other scenarios with SH, NH and HeVH patients. The number of veterinarians who always or sometimes used PPE and the amount of PPE used by participants increased as the health status of horses deteriorated. It is likely that veterinarians were making PPE choices according to their perceptions and assessments of the infectious risks involved. Risk perception and risk assessment were not the only factors determining PPE usage. Veterinarians in highly accessible and accessible ARIA regions were more likely to use PPE when examining a healthy horse. This may be because most of the highly to accessible ARIA regions were located in the known geographic distribution range of HeV in QLD; i.e., coastal areas between Far North QLD and South East QLD ( DAFF QLD, 2014 ). Veterinarians in less accessible, inland areas may have perceived the risk of being exposed to HeV as low or were less used to take HeV risks into consideration. The use of PPE by these veterinarians may have also been influenced by the different nature of their clientele. Horse owners from the racing industry were reported to react differently to the veterinary use of PPE than horse owners in rural areas ( Mendez et al., 2012b ). Rural clients often had different cost benefit expectations of veterinary service provided than other clients and/or viewed the systematic use of PPE by veterinarians negatively ( Mendez et al., 2012b ). Veterinarians who had graduated from a QLD university were also more likely to have a PPE usage score above the median score when examining a sick horse than participants who had graduated elsewhere. HeV first emerged in QLD and the majority of the outbreaks have since occurred in this state. Thus, QLD graduates may have received more formal and practical education about HeV during their undergraduate studies. This may have changed since 2011. However, if discrepancies were to remain, they potentially could put young graduates from other states at a higher risk of exposure to HeV. Early career veterinarians tend to have more than one employer within the first 2 years after graduation while some do not stay in the state they graduated from ( Heath, 2008 ). Therefore, all veterinary graduates from all Australian states should be equally made aware of potential occupational infectious risks relevant to all parts of the country. As such, undergraduate veterinary curricula should be consistent across all Australian universities. The usage of PPE also differed between participants who had attended an IC/HeV management training session in the previous 12 months; had access to a dedicated HeV management plan within their practice; had to deal with at least one potential case of HeV and those who had not. Practical HeV management experience in the field and through professional education as well as leadership in HeV management through planning within the practice may have been more effective in improving veterinarians’ usage of PPE when attending to equine patients than the broadly targeted government information campaigns. Alternatively, those who had already had experience with HeV or HeV management may have been more receptive to the government's campaigns and more motivated to implement the appropriate measures. Participants who rarely or never used PPE with healthy horses and had no experience with HeV or had not sought training or management planning may also have thought that the lack of clinical signs in a horse patient was likely to indicate a low risk of HeV exposure. However, experimentally infected horses have been shown to excrete viral particles at least 48 h before the onset of the first clinical signs, but this was not known until 2011 ( Marsh et al., 2011 ). Overall state of HeV management by private veterinarians HeV outbreaks have been occurring sporadically in QLD and Northern New South Wales since 1994 ( DAFF QLD, 2014 ). However, of the 49 outbreaks that have occurred to date, over a third happened in the winter of 2011 (17/49), and more than half of the 2011 outbreaks occurred in QLD (10/17) ( DAFF QLD, 2014 ). Despite this increase in HeV outbreaks there were no cases of human infection during this time. The uncharacteristic higher number of HeV outbreaks in 2011 may have increased the relevance of HeV risk for veterinarians in QLD who in turn adopted HeV management strategies more readily during this time. Their improved management of HeV may also be the result of a better understanding of the HeV related risks and HeV risk mitigation strategies by the veterinary profession; an improved support from government to veterinarians in the field as recommended by the Ombudsman in 2011 ( Clarke, 2011 , Queensland Government, 2011 ); and a better collaboration of government authorities with private veterinarians. However, since 2011 the number of outbreaks per year has greatly decreased and motivation to sustain HeV management strategies may have changed. This study has also shown that participants with some previous HeV experience or with pre-existing vested interest in HeV management were more likely to adopt and maintain HeV management strategies and attitudes. Hence, practical knowledge about HeV risk and risk mitigation appear to act as a stronger motivator than state wide education campaign promoted by Biosecurity QLD. This concurs with studies conducted in human healthcare settings where the role of education in the adoption of IC measures by health professional was not as strong as expected ( Ward, 2011 ). This should be taken into consideration when devising professional education programmes about the management of specific diseases to increase the effectiveness of these programmes. The introduction of a HeV vaccine for horses in 2012 ( Pallister et al., 2011a , Pallister et al., 2011b , Middleton et al., 2014 ) may also have since influenced HeV management by private veterinarians. Private veterinarians in QLD were very favourable to the HeV vaccine for horses prior to its introduction ( Mendez et al., 2013a ). The HeV vaccine has since been promoted as essential to the prevention of HeV in horses and their carer which may give a false sense of safety to veterinarians as not all horses in QLD and NWS have yet been vaccinated ( DPI NSW, 2013 ). The effect of the HeV vaccine release on the veterinary profession's commitment to IC for the management of HeV should be evaluated in the future. Representativeness, participation rate, limitations and strengths of study Of the 1604 eligible veterinarians, 200 (12.5%) completed the questions relevant to this part of the study and returned the questionnaire. However, not all participants answered all questions. Although 12.5% may seem a low level of response, this percentage does not represent the true response rate as the total number of private veterinarians providing equine services in QLD and therefore the true response rate could not be calculated. According to the 2009 president of the QLD division of the Australian Veterinary Association, the Equine Veterinarians Association counted 219 members which however did not include all equine veterinarians in the state of QLD (Dr, B. Pott, personal communication). Furthermore, a 2004 study estimated that equine veterinary services were provided by 12% of all Australian veterinarians ( Heath, 2004 ). Assuming these results were also representative of the veterinary population in QLD in 2011, the 200 veterinarians who participated in the present study may in fact represent a high proportion of the total number of veterinarians providing equine services in QLD. Another potential limiting factor to this study was that the private veterinary workforce in QLD had been under high scrutiny from the government in 2010 in relation to IC and HeV management ( WHS QLD, 2011b ). As a result, eligible veterinarians may have elected to decline participation in another investigative exercise into their practice; while those who chose to participate may have altered their responses in order to show a higher level of compliance with government recommendations in regards to IC/HeV management. Although it is difficult to gauge the validity of the results, the responses given by participating veterinarians in this study corroborate some of the findings from an exploratory qualitative study conducted by the authors in 2009–2010 ( Mendez et al., 2012a , Mendez et al., 2012b , Mendez et al., 2013b )."

"Conclusion Despite a marked increase in the number of HeV cases in 2011, there were no new human cases of HeV. This may indicate that QLD veterinarians were protecting themselves and/or managing HeV better than in the preceding years. The unusual epidemiology of HeV during this time may have served as a motivation boost for the veterinary profession and biosecurity authorities to improve HeV management. It also tested the newly developed HeV management strategies put in place by Biosecurity QLD from 2010 onwards: better flow of information and support in the field to veterinarians and horse owners. The results of this study seem to confirm that private veterinarians can better fulfil their biosecurity, public health and WHS roles, when dealing with emerging zoonoses such as HeV, when supported by the relevant authorities. This should be taken into consideration when devising strategies to manage future emerging zoonoses. However, despite the improved HeV management support provided by government authorities to the veterinary profession in QLD, HeV management strategies and behaviours were not found to be optimal across all participants and their practices, leaving some veterinarians at risk of exposure to HeV. The uptake of improved veterinary IC strategies by the private veterinarians may therefore depend upon other factors, such as risk perception, which should be further investigated. Moreover, until the HeV vaccine coverage of equine populations is consistently high across QLD and Northern NSW; IC, including the use of appropriate PPE, remains the best course of action to prevent human exposure to HeV. Therefore, HeV-related risk information and risk mitigation communication to veterinarians and horse owners need to remain a priority of the overall HeV management plan at the State and National levels. This should include regular updates about HeV to the veterinary profession, particularly advances in knowledge; education of horse owners; practical HeV management education to veterinarians; and the consistent education of undergraduate veterinary students Australia wide. The determinants of veterinary IC should also be further investigated in particular the role of undergraduate and professional education."

"Following the emergence of Hendra virus (HeV), private veterinarians have had to adopt additional infection control strategies to manage this zoonosis. Between 1994 and 2010, seven people became infected with HeV, four fatally. All infected people were at a higher risk of exposure from contact with horses as they were either veterinary personnel, assisting veterinarians, or working in the horse industry. The management of emerging zoonoses is best approached from a One Health perspective as it benefits biosecurity as well as a public health, including the health of those most at risk, in this case private veterinarians. In 2011 we conducted a cross-sectional study of private veterinarians registered in Queensland and providing veterinary services to horses. The aim of this study was to gauge if participants had adopted recommendations for improved infection control, including the use of personal protective equipment (PPE), and the development of HeV specific management strategies during the winter of 2011. A majority of participants worked in practices that had a formal HeV management plan, mostly based on the perusal of official guidelines and an HeV field kit. The use of PPE increased as the health status of an equine patient decreased, demonstrating that many participants evaluated the risk of exposure to HeV appropriately; while others remained at risk of HeV infection by not using the appropriate PPE even when attending a sick horse. This study took place after Biosecurity Queensland had sent a comprehensive package about HeV management to all private veterinarians working in Queensland. However, those who had previous HeV experience through the management of suspected cases or had attended a HeV specific professional education programme in the previous 12 months were more likely to use PPE than those who had not. This may indicate that for private veterinarians in Queensland personal experience and face-to-face professional education sessions may be more effective in the improvement of HeV management than passive education via information packages. The role of different education pathways in the sustainable adoption of veterinary infection control measures should be further investigated. Keywords"

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"Acknowledgements The authors would like to thank all veterinarians who participated and Susan Reilly who provided data management support. Funding for this project was provided by the Anton Breinl Centre for Public Health and Tropical Medicine through its Wildlife Services Fund; and by 10.13039/501100001792 James Cook University through a Higher Degree Research scholarship granted to D. Mendez."

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"2023-06-06 23:35:25"

"Prev Vet Med. 2014 Nov 1; 117(1):40-51"

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"Introduction The situation of the world for the people is very risky to spend the peaceful life due to the spreading of the COVID-19. The COVID-19 is viral disease, a pandemic and the world health organization (WHO) declared an emergency situation due the spreading of COVID-19. In the end of 2019, some cases reported as same symptoms in the Wuhan city, province Hubei, China, after the diagnosing of these cases reported as novel coronavirus (COVID-19). This deadly virus has infected the entire world and many people have died as a result of this insuperable virus. The name “coronavirus” comes from the Latin word “corona” which means a “crown, circle of light or nimbus”. This virus influences immediately to your lungs. It has comparable symptoms as influenza and pneumonia. In the beginning, various of those infected worked or shopped at a wholesale seafood market in Wuhan, China. After that it radiates universally through import, export, travelling and social contacting of infected people. The Fig. 1 represents the world wide confirmed cases till May 4, 2020. Several researchers investigated and developed different methods for addressing obstacles to medical and decision-making. In practical decision making, there are a great quantity of uncertainties, imprecise and vague information, whose representations and managements are always the central issues. Health professionals and healthcare administrators are working to reduce clinical and maintenance costs for the prevention and management of corona disease. Expenditure and need for health care are both growing fast. Health care practitioners, administrators and other sectors collectively perform a range of healthcare management techniques with the goal of facilitating effective disease prevention approaches using scarce resources. Such principles are used to build a decision-making model using a number of parameters and alternatives (Cromwell et al. 2015 ). The purpose of the multiple criteria decision making (MCDM) frameworks is to prepared an appropriate decisions at different levels of health care, such as operational, methodical, and functional. There may be an ideal solution to any difficult decision-making problem, but it is a difficult task to find such a method. In particular, management decisions are taken by managers or senior management to grow and maintain the organization. In fact, there are contradictions in strategic decisions, possible synergies between different options, and uncertainty in the final result. When strategic decisions are taken, the company shall agree on tactical and operational planning decisions. Strategic, tactical, and operational planning are grouped together as a taxonomy of health planning (Kumar et al. 2017 ). Disease prevention and control approaches include multiple management roles like as facility preparation, organization and decision making. MCDM problems with spherical fuzzy environment took much attention to the real-life problems where the goal is associated for selecting the best alternative in contrast to the nite values under the different criteria where the evaluation terms are SFNs given by decision experts (DEs). However, in order to process the ambiguity /imprecision in the data, theories like as fuzzy set (FS) (Zadeh 1965 ), intuitionistic FS (IFS) (Attanassov 1986 ), picture FS (Cuong and Kreinovich 2013 ), spherical FS (Ashraf and Abdullah 2019 ), are applied widely. Presently, decision-making is a hot topic in the field of research which includes the following three main steps: To describe the information, collect the data on an appropriate scale. Obtain the totally preference value of the object by assigning the various attribute values. Rank the objects in a transparent process to get the suitable alternative(s). Therefore, the intention of the present research is to describe a group decision making method to resolve the multicriteria group decision making (MCGDM) problems for SFSs with robust generalized TOPSIS-COPRAS approach based on the spherical fuzzy information. The novelty of fuzzy set firstly defined by Zadeh ( 1965 ) to use non-statistical and vague phenomena. Since the inception, the theory of FS became a more interesting research area, e.g., image processing, data mining, engineering, medical sciences, clustering, statistical information theory and information technology. Since FSs assign only a crisp membership function of an element to show the double conflicting states, one is support and other is disagree. Thus, fuzzy set theory faces the limitation to show the negative state. To avoid this limitation, Atanassov (Attanassov 1986 ) developed the idea of intuitionistic fuzzy sets (IFSs) theory based on the notion of fuzzy set (FS) by Zadeh. The application of IFSs have investigated by many authors (Mendel et al. 2019a , b ; Mendel 2019b ). Atanassov Atanassov ( 2018a , 2018b , 2015 ) presented the dfferent decision making techniques to tackle the uncertainty in real life decision making problems. Sotirov et al. ( 2018 ) introduced the hybrid approach for modular neural network design using intercriteria analysis and intuitionistic fuzzy logic. Sotirov et al. ( 2016 ); Castillo et al. ( 2015 ) proposed the novel modular neural network preprocessing procedure with intuitionistic fuzzy intercriteria analysis method to tackle the uncertainty in real life DMPs. Although, IFS based models have been successfully implemented in different areas since its appearance, but there are practical situations in real-world which cannot be represented by the traditional IFSs. Recently, (Cuong and Kreinovich 2013 ) filled these gaps by introducing the neutral membership in Atanassov’s IFS theory. Picture fuzzy set (PFS) in a finite fixed set is written as where with condition that . Basically, PFSs can precisely describe a human views, including more responses, such as: “yes”, “abstain”, “no” and “refusal”. Many researcher (Ashraf et al. 2019e , f ; Khan et al. 2019a , b , c ; Wei 2017 ; Zeng et al. 2019 ) contributed to the picture FS. Since the introduction of IFS, the theories and applications of IFS have been studied comprehensively, including its’ applications in DMPs. These researches are very appropriate to tackle DMPs under PFS environment only owing to the condition . However, in practical DMPs, the decision makers provides evaluation value in the form of , but it may be not satisfy the condition and beyond the upper bound 1. Aiming at this limitation which PFN can not handle, (Ashraf and Abdullah 2019 ) established a new concept of spherical fuzzy (SF) set to handle with this situation. SFS is an extension of PFS by slackening the condition . We must also note that the acceptable spherical fuzzy space increases, thus providing more freedom for observers to express their belief in supporting membership. Therefore, SFSs express more extensive fuzzy information; Whilst, SFSs are more maneuverable and more appropriate for dealing with uncertainties information. Several researchers have done quite valuable contributions in the expansion of SF set and its approach to different fields, their results shows the great success of SF set in theoretical and technical aspects. As aggregation operators have a strong role to play in decision-making problems (DMPs), several researchers have done quite valuable contributions to introduce aggregation operators for SF set. Spherical aggregation operators based on algebraic norms (Ashraf et al. 2019a ) dealing with uncertainty and inaccurate information in DMPs. SF set the representation of SF norms (Ashraf et al. 2019b ) and TOPSIS methodology introduced for SF information. SF Dombi aggregation operators based on Dombi norm are introduced in Ashraf et al. ( 2019c ). SF Logarithmic aggregation operators based on entropy are proposed in Jin et al. ( 2019a ). Linguistic SF aggregation operators are presented in Jin et al. ( 2019b ) for SF information to tackle the uncertainty in DMPs. Cao ( 2019 ) proposed the spherical linguistic Muirhead mean operators and discussed their application in group DMP. GRA methodology based on spherical linguistic fuzzy Choquet integral is proposed (Ashraf et al. 2018 ) for SF information. Cosine similarity measures are presented in Rafiq et al. ( 2019 ) to discussed the application in DMPs. Application of SF distance measures are discussed in Ashraf et al. ( 2019d ) to determined the child development influence environmental factors using SF information. In Zeng et al. ( 2019 ) proposed the TOPSIS approach based on SF rough Set and discussed their application in DMPs. Gündoğdu et al. ( 2020b ) presented the TOPSIS methodology using SF information and discussed their real life application in DMPs. Gündoğdu and Kahraman ( 2020c ) introduced the QFD method and also presented its application to the linear delta robot technology development problem. Gündoğdu ( 2020a ); Gündoğdu and Kahraman ( 2019 ) exted the concept of spherical fuzzy set to interval-valued fuzzy set and presented the decision making methodology to tackle in uncertainty in DMPs. Khan et al. ( 2020a ) introduced the distance and similarity measures for spherical fuzzy sets and discussed their applications in selecting mega projects. Ashraf et al. ( 2020g ) proposed the symmetric sum based aggregation operators for spherical fuzzy information and discussed their application in multi-attribute group decision-making problem. Ashraf et al. ( 2020h ) presented the decision making technique using sine function and Barukab (Barukab et al. 2019 ) introduced new approach to fuzzy TOPSIS method based on entropy measure under spherical fuzzy information. Just like these DM methods, we have the most fruitful method called TOPSIS method, which was introduced in 1981, by Hwang and Yoon ( 1981 ). The abbreviation, TOPSIS stands for “technique for order preference by similarity to the ideal solution. This method was developed later by many authors. The high flexibility of the TOPSIS concept allows us to add additional extensions to make the best choices in different situations. Practically, TOPSIS and its modifications are used to solve many theoretical and real-world problems (Boran et al. 2009 ; Chen 2000 ; Nag and Helal 2016 ; Wang and Elhag 2006 ; Wang et al. 2018 ). In complex decision making, where the results can be easily evaluated by using TOPSIS method, contains a lot of qualitative information. The decision makers have limited attention and information processing skills. The TOPSIS method is a practical and useful technique for ranking and selection of alternatives. Complex Proportional Assessment (COPRAS) (Zavadskas and Kaklauskas 1996 ) methodology proposed by Zavadskas and Kaklauskas in 1996, which is most effectively and commonly used technique to deal with the uncertainty in DMPs. It is used to evaluate alternatives dependent on several criteria by applying the corresponding weights of parameters and the degree of usefulness of alternatives. Choosing the appropriate alternative is achieved by focusing at the ideal and anti-ideal solutions. COPRAS claims that the importance and usefulness features under investigation are directly and proportionately dependent on a set of criteria that describes alternatives efficiently and on the criteria’s values and weights. COPRAS has many benefits, such as less processing time, a very easy and straightforward method of computing etc, over other MCDM methods such as EVAMIX, VIKOR and AHP. With respect to the advantages of SF set in describing uncertain information, also, regardless of the motivation and inspiration of all the above debate, we enlist the main objectives of the article: Article main objective to provides a new strategy to SF set through emergency group decision making problem (GDMP) for control and prevent the COVID-19 effectively. In this paper, a new methodology based on TOPSIS approach hybrid with the COPRAS, which can deal much more uncertainties in the form of spherical fuzzy sets. Note that, in comparisons with the classic fuzzy sets, spherical fuzzy set has more capability to deal the different situations more successfully. In fact, these sets consider opinions of DMs better than classic fuzzy sets. That is why, to use advantages and flexibility of the SF sets, the introduced technique is established under these sets to discourse the uncertainty of real-life in better way. We design an algorithm to tackle emergency decision-making problem of COVID-19. We shall collect the exact data disaster during the COVD-19 and then construct the mathematical model of emergency decision support systems for COVD-19 under generalized structure of spherical fuzzy sets and compare our propose technique with existing techniques to shows the validity and effectiveness of the proposed methodology. To achieve the list of goals the structure of the paper is arranged as follows: In Sect. 2 , some basic concepts are introduced. In Sect. 3 , proposed the different types of distance between SF numbers. Section 4 , gave the main contribution of the paper, introduced the TOPSIS-COPRAS technique to deal with the uncertainty in DMP using SF information. Section 5 , propose the numerical case study of outbreak of coronavirus as an emergency decision support problem to demonstrate the applicability and reliability of the proposed technique. Section 6 presents the comparison analysis to shows the applicability of the proposed methodology and concluded remarks are discussed in Sect. 7 ."

"Proposed methodology In this segment, we proposed the methodology to deal with uncertainty and inaccurate information in the form of SFSs in DMPs. The proposed methodology has following steps: Step-1 Data Collection Judgements of specialists’ decision maker (DM) experts on assessments criteria for every activity and each criterion weights are assembled in the shape of initial decision matrixes. At primary, the matrix constructed on ideas of kth DM is computed as below: where, denotes the activities, denotes the criteria, respectively, and represents the specialists’ decision makers. Then, the spherical fuzzy matrix constructed on ideas of kth DM is computed as follows: where, denotes the numbers of paths (alternatives). Step-2 Calculation Of DMs Weights Each specialists’ decision maker give specified weight to decision matrix. In this step, we calculate the weights of the decision matrices by utilizing the closeness to average ideal solution and maximum distance from positive and negative ideal solutions. Step-2(a) In this step, utilizing (Yue 2011 ) methodology to find the average , left negative and right negative ideal solutions as follows where with and where with and where with and Step-2(b) To measure decision level of each DM, we find the distance between each individual decision matrix with average ideal matrix left negative ideal solution and right negative ideal solution . Consider that the Euclidean distance is the most widely used tool to measure the separation of two objects in practical applications, we utilize it to measure the separation between with and as follows. Step-2(c) Proposed the final closedness coefficient value of each DM is calculated as where Step-2(d) Final weights of each DM is obtained as Step-3 Aggregated matrix is obtained by using spherical fuzzy weighted averaging operator Step-4 Aggregated spherical matrix for all the possible paths is constructed by using the addition rules of spherical fuzzy set as follows Step-5 Positive ideal and negative ideal solutions are calculated as and Step-6 Calculate the Euclidean distance of aggregated spherical fuzzy information from the positive and negative ideal solutions as follows and Step-7 Closeness relation to ideal solutions are calculated as follows To ranked the set of paths (alternatives) by preference according to the descending order of Means highest will be our finest path (alternative). Flow chart of the proposed technique is given in Fig. 2 :"

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"Conclusion The novel 2019 Coronavirus, SARS-CoV-2 (COVID-19), originated in the city of Wuhan in the People’s Republic of China’s Hubei province towards the end of 2019 and has spread very quickly in a very short time to the world. This article aimed to analyze the pandemic trajectory using mathematical modeling based on the information used by fuzzy decision making methodology to select the best alternative using critical path strategy. Spherical fuzzy set plays a vital role in solving emergency decision making in the emergency situation of COVID-19, as they can optimal describe a preference when there is vague or uncertain information. In this study, a new integrated TOPSIS-COPRAS approach is established to handle emergency MCGDM problems with unknown weight information. The presented approach simultaneously considers a DMs’ limiting rationality and interdependence among criteria. The objective weight vectors are obtained by using the distance measure and were combined with subjective weights in the spherical fuzzy MCGDM model. Moreover, the operating of the proposed method is thoroughly explained with the assistance of a numerical example on the basis of the TOPSIS-COPRAS method. We testified the effectiveness and rationality of the proposed MCGDM approach, its output is compared with other MCGDM problems to make a comparison. The proposed MCGDM approach can also be used to other complicated problems like risk evaluation, emerging technology, uncertain decision-making, project installation, site selection etc. The approach proposed in this paper will be extended in future research to other ambiguous fields, such as linguistic term sets, probabilistic linguistic term sets, hesitant fuzzy sets etc. The suggested approach can also be extended to other fields, such as medical diagnosis of nutrition, sustainable choice of suppliers, pattern recognition and so on. We will also try to extend this work for interval valued spherical fuzzy environments."

"Communicated by Valentina E. Balas. The control of spreading of COVID-19 in emergency situation the entire world is a challenge, and therefore, the aim of this study was to propose a spherical intelligent fuzzy decision model for control and diagnosis of COVID-19. The emergency event is known to have aspects of short time and data, harmfulness, and ambiguity, and policy makers are often rationally bounded under uncertainty and threat. There are some classic approaches for representing and explaining the complexity and vagueness of the information. The effective tool to describe and reduce the uncertainty in data information is fuzzy set and their extension. Therefore, we used fuzzy logic to develop fuzzy mathematical model for control of transmission and spreading of COVID19. The fuzzy control of early transmission and spreading of coronavirus by fuzzy mathematical model will be very effective. The proposed research work is on fuzzy mathematical model of intelligent decision systems under the spherical fuzzy information. In the proposed work, we will develop a newly and generalized technique for COVID19 based on the technique for order of preference by similarity to ideal solution (TOPSIS) and complex proportional assessment (COPRAS) methods under spherical fuzzy environment. Finally, an illustrative the emergency situation of COVID-19 is given for demonstrating the effectiveness of the suggested method, along with a sensitivity analysis and comparative analysis, showing the feasibility and reliability of its results. Keywords"

"Preliminaries In this section, for better understanding of the spherical fuzzy sets, some related basic concepts will be briefly reviewed. Definition 1 Zadeh ( 1965 ) A fuzzy set in fixed set is described as where called positive membership grade. By we mean that for each Clearly if and Utilizing (Zadeh 1965 ), proposed min–max system to define basic operational laws as follows: where and Definition 2 (Ashraf and Abdullah 2019 )A spherical fuzzy set in fixed set is described as where positive membership, neutral membership and negative membership grades, respectively. In addition, it is necessary to for each . To what follows, we symbolize the collection of all spherical fuzzy sets in by . For convenience, the spherical fuzzy number (SFN) is symbolized by the triplet Let Ashraf and Abdullah ( 2019 ) defined the following notions: if and for each Clearly if and where and Definition 3 (Ashraf and Abdullah 2019 )Let and with Then, the operational rules are as follows: Definition 4 Ashraf et al. ( 2019a )Let and be a mapping defined as Then, by operational laws of SFNs, we obtained spherical fuzzy weighted averaging operator as where the weight vector of with and is Definition 5 Ashraf et al. ( 2019a ) Let and be a mapping defined as Then, by operational laws of SFNs, we obtained spherical fuzzy weighted geometric operator as Where the weight vector of with and is Distance of spherical fuzzy sets Definition 6 Let and Then maximum distance is defined as Definition 7 Let and Then minimum distance is defined as Definition 8 Let and . Then Hamming distance is defined as Definition 9 Let and . Then Euclidean distance is defined as Definition 10 Let and . Then normalized Hamming distance is defined as Definition 11 Let and . Then normalized Euclidean distance is defined as Application To study the prevention and control of COVID-19, we have developed a novel hybrid methodology for selecting the best alternatives using a critical path strategy that will help to choose the best path to overcome this deadly disease. Case Study: To demonstrate the applicability and validity of the proposed methods, we extant a real case study about an emergency caused by an outbreak of novel Coronavirus disease (COVID-19) pandemic that occurred in China. Since 19 December 2020, in Wuhan, China, there have been several unidentified cases of pneumonia with cough, dyspnea, exhaustion and fever as the major symptoms reported in a short time. The Chinese health officials and CDC immediately identified the pathogen of these cases as a new form of coronavirus which was called COVID-19 by the World Health Organization (WHO) on 10 Janvary-20 (World 2020 ). The Chinese government’s information department held a press conference on pneumonia prevention and control of new coronavirus infections on January 22, 2020. The same day, a strategy for the prevention and control of pneumonitis of new coronavirus infection was announced by the People’s Republic of China, along with COVID-19 epidemic research, sample collection and testing, monitoring and management of close contacts, and public propaganda, education and risk communication (Shen et al. 2020 ). As of May 4, 2020, more than 3 442 234 confirmed cases and 239 740 confirmed deaths are reported in 215 Countries, areas or territories. The infected cases graph are as follows in Fig. 3 : In such emergency situation, it is essential to provide an efficient way in emergency response for avoiding additional losses and to save the lives of the people. Preventive and mitigation measures are key in both health care and community settings. Due to such an emergency decision, the health experts have to make an immediate response, urgently rescue to control the situation efficiently and stop it from more deaths. The panel of three experts ratings on the set of criteria are collected and illustrated for each activity shown in Tables 1 , 2 . Step-1 Decision makers activities information computed in spherical fuzzy sets using Table 2 : Step-2(a) Utilizing (Yue 2011 ) methodology to find the average , left negative and right negative ideal solutions are given as follows Step-2(b) We find the and by using formulas of Step-2(b). Step-2(c) The final closedness coefficient values are obtained using Eq. 4.8 and Step-2(d) Weights using Eq. 4.9 are follows as Step-3 Calculate the aggregated matrix by using spherical fuzzy weighted averaging operator defined in Eq. 4.10 in Table 3 (a), (b). There is a panel of experts to determined the critical path (given in (Fig. 4 )) for prevent and control of COVID-19 with respect to the following criteria’s: Step-4 Calculated aggregated spherical matrix for paths by using addition rule of spherical fuzzy set are evaluated in Table 4 (a), (b). Step-5 Calculate the Positive ideal and negative ideal solution by using Eq. 4.11 and Eq. 4.12 . and Step-6 Calculate the Euclidean distance of aggregated spherical fuzzy information from the positive and negative ideal solutions by using Eqs. 4.13 and 4.14 as follows in Table 5 (a): Step-7 Calculate the closeness relation value by using Eq. 4.15 . and Final ranking are as follows in Table 5 (b): Comparison analysis In the following, we will demonstrate the effectiveness and advantages of proposed operators by comparing with the existing methods. The final ranks of alternatives (paths) are similar. In view of this the approach proposed is valid. Table 6 displays the final results of the proposed approach and TOPSIS process. In addition, comparisons of the current approach with the preceding studies to clearly clarify the implications of the proposed approach are displayed in Table 7 . Additionally, the comparisons between two forms of fuzzy sets are shown in Table 8 . As can be shown, under IFSs and PFSs environments, the essential path of the project network remains the same; however, other ranks (project paths) have been modified. With all of this in view, the SF sets may understand uncertainty better than the existing fuzzy set structure. The critical path of the network is identified correctly by using the proposed methodology. As a result, project scheduling and planning may be closely related to reality. In fact, in an uncertain environment, the critical path of the projects and the degree of criticality of each path are specified. Method flexibility with various input and outputs The proposed methodology are flexible, and can be efficiently used for various input and output circumstances. Because of the different score functions and its generalization, the ranking of the proposed technique seems to differ little. This model is more efficient than most because, in decision-making methods, spherical fuzzy set increases grade space and can variate according to the emergency situations. Superiority of suggested methodology and comparison with other frameworks Fuzzy set, intuitionistic FS, picture FS have some space limitation on their grades. Spherical FS fills this gap in the literature and offers significant space than FS, intuitionistic FS, picture FS. The suggested framework enhances existing approaches and the decision-maker can choose the grades freely by using the condition Limitations The limitation of this analysis is that the developed model determines the best alternative in a single setting based on the input of considered experts."

"Acknowledgements This work was supported by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (D-579-611-1441). The authors, therefore, gratefully acknowledge DSR technical and financial support. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors."

"NO-CC CODE"

"no"

"2023-06-06 23:35:14"

"Soft comput. 2023 Oct 1; 27(3):1809-1825"

"oa_package/3b/f9/PMC7529095.tar.gz"

"PMC7825904"

"33608162"

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"Sr. Director: La aspergilosis pulmonar invasiva puede complicar algunas infecciones víricas, como la gripe, y comienza a evidenciarse como factor de mal pronóstico en pacientes coinfectados con neumonía por SARS-CoV-2. Comentamos el caso de un varón de 67 años, con enfermedad renal crónica secundaria a glomerulonefritis focal y segmentaria, en hemodiálisis. En junio de 2020 recibe trasplante renal, realizándose inducción con basiliximab y tratamiento con tacrolimus, micofenolato y esteroides. Además asocia profilaxis con cotrimoxazol y valganciclovir. Treinta días postrasplante se diagnostica por SARS-CoV-2. Se reduce la dosis de inmunosupresores y se inician azitromicina e hidroxicloroquina. El día +14 ingresa por fiebre e insuficiencia respiratoria. En la radiografía de tórax presenta infiltrados bilaterales y en la analítica presenta creatinina 1,5 mg/dL, PCR 72 mg/L, Hb 10,6 g/dL, linfocitos 340/ μL, dímero D 1,021 ng/mL e interleucina-6 31,9 pg/L. Al ingreso se suspende micofenolato y se inicia tratamiento con dexametosona + remdesevir + ceftriaxona + heparina profiláctica. Por criterios de gravedad se administra tocilizumab al tercer día de ingreso y se suspende tacrólimus el quinto día por mala evolución y por niveles supraterapéuticos. El día +7 ingresa en la UCI por deterioro del nivel de consciencia e insuficiencia respiratoria que precisa ventilación mecánica. Se amplía cobertura con meropenem, amikacina, linezolid y voriconazol, manteniendo valganciclovir y soltrim profilácticos. Además de la persistencia de PCR positiva para SARS-CoV-2, en el broncoaspirado de rutina se halló Aspergillus fumigatus y el galactomanano en suero fue de 4,5. Dada la persistencia de niveles elevados de tacrolimus se sustituye voriconazol por isavuconazol intravenoso. Tras 13 días de ingreso el paciente presenta mala evolución con hemorragia cerebral masiva y fallece ese mismo día. El Registro COVID-19 de la Sociedad Española de Nefrología (SEN) en noviembre 2020 reporta 2.474 pacientes en tratamiento sustitutivo renal, de los cuales un 37% son trasplantados renales 1 . Esta población es considerada de mayor riesgo debido a su estado de inmunosupresión y al contacto frecuente con centros sanitarios 2 . Los pacientes gravemente enfermos de COVID-19 presentan concentraciones más elevadas de citocinas proinflamatorias (IL-1, IL-2, IL-6 y factor de necrosis tumoral alfa) y antiinflamatorias (IL-4 e IL-10), con menor expresión de interferón-gamma y tienen un número más bajo de células CD4 y CD8 3 . Por ello, el riesgo de padecer coinfecciones fúngicas es mayor 4 . De hecho, se ha descrito una incidencia de aspergillosis invasiva de hasta un 0,65% dentro del primer año en trasplantados renales, con una tasa de mortalidad de hasta un 39% en las primeras 12 semanas 5 . A pesar del alto número de casos reportados de COVID-19, su asociación con aspergilosis invasiva ha sido poco descrita. El grupo europeo EORTC/MSG concluye que el diagnóstico de aspergillosis pulmonar asociada con la COVID-19 (APAC) constituye un reto, ya que las características radiológicas de la lesión invasiva fúngica se superponen a las alteraciones ya existentes por la neumonía viral por SARS-CoV-2 6 , 7 . Además, el elevado riesgo de generación de aerosoles limita en pacientes con COVID-19 la obtención de muestras respiratorias (broncoaspirado o lavado bronquioalveolar), por lo que el diagnóstico en muchas ocasiones se basa en el antígeno galactomanano en suero, considerado positivo un índice > 0,7 8 . El número de especies de Aspergillus spp. es muy numeroso, pero Aspergillus fumigatus complex es el agente etiológico más frecuente. El tratamiento de elección es el voriconazol. En nuestro caso se cambió a isavuconazol debido a la imposibilidad de la vía oral, su menor influencia en la actividad del CYP3A4 (el paciente tenía niveles supraterapéuticos a pesar de haber suspendido tacrolimus) y su mayor capacidad de atravesar la barrera hematoencefálica 9 . Sin embargo, es cada vez más frecuente la resistencia del Aspergillus fumigatus a los azoles. Algunos autores recomiendan evitar monoterapia y usar tratamiento combinado con equinocandinas o anfotericina B liposomal si existe sospecha de resistencia o mala evolución, realizando una identificación molecular 10 . Desafortunadamente la susceptibilidad antifúngica para el Aspergillus spp. no está disponible en todos los laboratorios o puede demorar mucho tiempo, por lo que es posible que la tasa de resistencia a azoles en nuestro medio esté infraestimada. En conclusión, la coinfección entre SARS-CoV-2 y micosis invasoras en pacientes inmunosuprimidos probablemente sea mayor de la descrita en la literatura. Por este motivo, y dadas las limitaciones del diagnóstico, la presencia de marcadores fúngicos debería aconsejar la instauración precoz de tratamiento."

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"NO-CC CODE"

"no"

"2023-06-06 23:35:03"

"Nefrologia. 2022 Jan 23 May-June; 42(3):359-360"

"oa_package/3d/7b/PMC7825904.tar.gz"

"PMC7856850"

"33551675"

"Introduction The coronavirus (COVID-19) has unfolded very swiftly throughout India and many other countries inflicting acute infectious pneumonia to break out (Bao et al. 2020 ). Staying at home is only the solution that can restrict the spreading of this disease. However, long stay at home can increase the sedentary activities (Owen et al. 2010 ) that lead to inactiveness. This inactiveness leads to anxiousness and unhappiness, and negative consequences on the fitness of human beings. Also, workers are subjected to a high level of musculoskeletal disorder (MSD) risk due to awkward postures in working for more extended hours during homestay. The different types of MSDs are most responsible reasons for losses in productive working time (Occupational Safety and Health Administration 2013 ). The association between MSDs and computer use has been made a public health concern since the mid-1980s when computer usage in working environments increased dramatically (Hopkins 1990 ). According to the U.S. Census Bureau report, 120 million American households (75% of population) had personal computers with internet in 2012, which increased 35% from 2001 (U. S. Census Bureau 2012 ). The use of handheld devices (HHDs) and internet users is also multiplying, as increase of 5.3% in the internet users was observed from 2018 to 2019, with a total user of 4.1 billion worldwide (International Telecommunication Union 2019 ). Also, it is clear from the report that the numbers of personal computers with internet access have been decreased in recent years. It shows the popularity of portable HHDs (i.e. smart phones, tablets, etc.), these devices enable the users to work anywhere and anytime (Saito et al. 1997 ; Moffet et al. 2002 ), which generates various work-related disorders specially MSDs. The generation of these disorders initiates due to various work-related risk factors, which are characterized in the following categories: i.e. physical factor (PF), psychosocial factor (PSF), and individual factor (IF) with their subfactors (Janwantanakul et al. 2012 ). Hence, it appears imperative to explore the literature related to MSDs in HHD users and earlier soft computing tools used for decision-making. The particulars of the remaining sections of this paper are described as: Sect. 2 discusses the relevant literature in this field. Section 3 represents the listing of primary factors and subfactors of risks which might result in the inception of MSDs indicated by the previous researches and decision-makers. Section 4 comprised of methodology to perform the current research. It includes the description related to implementation of integrated multi-criteria decision-making (MCDM) approach (BWM and VIKOR techniques). Section 4 describes the outcomes of the proposed integrated approach, and a comparison of current research outcomes with available literature. Finally, the last section exhibits conclusion, limitations, and future research directions based on the outcomes of current research."

"Methodology A three-stage methodology has been used in the current research (Fig. 2 ). The objective of using this three-stage procedure is to identify the risk level of MSDs among HHD users. The first stage includes the risk factors identification based on a previously published literature and suggestions of expert team. The second stage involves the priority building and relative importance (weightage) calculation of these risk factors by using the BWM technique. The third stage uses VIKOR technique for ranking the best alternative among seven types of HHD users with respect to priority and relative importance identified using BWM technique. BWM technique Rezaei ( 2015 ) established an efficient MCDM approach named BWM. Herein process, the decision-maker selects the best and worst factor/subfactor from the developed list of factors/subfactors of risks. The most significant and least favourable factors/subfactors are termed as the best and worst factors/subfactors, respectively. Decision-makers then do a relative comparison of the best factor with other factors, and other factors with the worst factor. This comparative analysis generates two pairs of comparison in vector form, which helps to find out the weights of the factors. Determination of the optimal weight of the factors/subfactors of risk is solved by a linear programming model for optimized outcomes. Implementation methodologies of the BWM are explained very well in the previous works (Rezaei 2015 , 2016 ; Gupta 2018 ; Khan et al. 2019 ). However, the BWM steps used in the current research are described below: Step I Discovery of all the decision criterion/factors and subfactors of risks is essential for decision matrix preparation. On the basis of decision-makers choice, the best and the worst factors/subfactors of risk are designated. Step II A pairwise evaluation matrix among the best risk factor/subfactor and all other factors/subfactors of risks is developed using a scale of 1 to 9 as given in Table 1 . This process is used for obtaining a preference of the best risk factor/subfactor over the others. The best from others (BFO) vector is defined as given in Eq. ( 1 ): where vector represents the preference of the best risk factor/subfactor (B) over the other factors/subfactors of risks ( ) and the self-preference defined as: . Step III The factors/subfactors of risks are now compared with the worst risk factor/subfactor in the form of pairs, which helps to generate the others to worst (OTW) evaluation matrix in the similar way as defined in step II. The vector matrix of OTW is represented as below: where vector represents the preference of the other factors/subfactors of risks ( ) over the worst factor/subfactor ( W ) and the self-preference is . Step IV. For the calculation of the optimum weights ( ), the absolute maximum modifications for all j are minimalized. The mathematical likeness (objective function) of this modification is written as: Objective function: . subject to: Equation ( 3 ) can be converted to the linear programming problem for determining the optimal weights. subject to: Equation ( 4 ) is solved for best result for finding the optimum weights ( ). Similarly, results of Eq. ( 4 ) delivers the optimum value of the consistency/reliability ratio . The assessed value of specifies the reliability of the evaluations made. The close to zero value of represents the high level of reliability in outcomes. VIKOR technique VIKOR technique is a passive ranking method (Opricovic 1998 ) and is used frequently where dissimilar contradictory measures are present. It generates a passive result based on “closeness to ideal solution and mutual agreement through concessions”. The VIKOR technique was extensively used by many investigators to get ranking of alternatives in the ergonomics design researches (Mohanty et al. 2018 ; Alsalem et al. 2019 ). The necessary steps of VIKOR technique employed in the current research are presented underneath: Step I Pairwise decision matrix generated for every alternative with respect to each subfactors of risks using linguistic scale of 1 to 5 is given in Table 1 . Step II The average decision matrix is processed using Eq. ( 5 ). Step III This step is used to calculate best values and the worst values from the average decision matrix of all alternatives. where denotes the positive best outcome and signifies the negative best outcome for the b th characteristic ( ) in the average decision matrix. Step IV The weighted and normalized Manhattan distance or Utility measure ( S a ) and weighted and normalized Chebyshev distance or Regret measure ( R a ) values are calculated using Eqs. ( 6 ) and ( 7 ). where S a denotes the distance of a th alternative ( ) from positive best outcome and R a denotes the distance of a th alternative from negative best outcome and W b represents the weights of subfactors of risks gained from BWM technique. Step V In this step, the scores for closeness coefficients or VIKOR index ( Q a ) are calculated using Eq. ( 8 ) and ( 9 ). where represents the weightage of supreme effectiveness and is assumed to 0.5 in the current research. Step VI In the last step, minimum score of Q a is ranked first among the all alternatives based on the two situations below: Situation 1: is selected if where is the alternative that has achieved second rank in the investigation and n is the total number of alternatives. Situation 2: also attains first rank conferring to both and values."

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"Conclusion, limitations and future scope Conclusion MSD is prominent health issue which forces workers to away from the work. Organizations are forced to disburse a larger amount of money to the workers experiencing MSDs in contradiction of their compensation claims. MSDs harmfully disturb workers’ health and also affect the work steering to significant loss of productivity and efficiency. Numerous risk factors have been testified for MSD generation, out of which some are prominent risk factors and few are risky. In the current research, an approach was proposed to decide priority and optimal weightages of the MSD risk factors using BWM. Also, the alternatives with respect to risk factors were ranked for selection of risky device users among all HHD users. The outcomes of the current research provided the following conclusions: Out of three primary types of MSD risk factors, physical factors (PF) are the prominent followed by psychosocial factors (PSF) and individual factors (IF). The rank of prominence of the different subfactors of the physical factors (PF) for MSDs is found as DW > PO > PD > FE. The various subfactors of the psychosocial factors (PSF) follow the order as JS > RW > JSA > TAT in increasing MSDs. The ranking of the subfactors in the individual factor (IF) category is found in the order of PA > AG > OB > SM > GE. The ranking of seven type of HHD users is as A2 > A1 > A4 > A3 > A6 > A5 > A7, and the computer professionals are at higher risk among seven type of HHD users. Limitations of the present research Similar to previous investigations, the current research has also particular limitations which are described as: The linear or combined interacting influences of the different risk factors are not prioritized in our research, though such connections have been conveyed to source of MSDs. Our research uses a small group of four decision-makers for all judgements taken in both technique and does not include any decision-maker form industry. Future research directions The possibility for future work is always present in a research. In view of above explained restrictions, it is recommended that upcoming works might be performed to prioritize linear or combined interactions of the risk factors using suitable soft computing approaches with aggregation of factors as utilized in previous researches (Jana et al. 2019 , 2020 ). Additionally, a comparatively bigger crowd of decision-makers from both industry and academia may be absorbed for gathering rating data. This collected data may be further analysed by using various evolutionary algorithms (Monte Carlo simulation, stochastic modelling, neural network, etc.) for building the prediction models based on various risk factors. These type of approaches were provided the effective results in the previous researches (Yi et al. 2018 ; Castillo and Melin 2020 ; Dansana et al. 2020 ; Kannan et al. 2020 ; Melin et al. 2020a , b ). The compulsory input (on the linguistic scale) from all decision-makers may be gathered independently for optimum weight computation of the risk factors using BWM."

"Communicated by V. E. Balas. In work-from-home (WFH) situation due to coronavirus (COVID-19) pandemic, the handheld device (HHD) users work in awkward postures for longer hours because of unavailability of ergonomically designed workstations. This problem results in different type of musculoskeletal disorders (MSDs) among the HHD users. An integrated multi-criteria decision-making approach was offered for identifying the risk level of MSDs among HHD users. A case example implemented the proposed approach in which, firstly, the best–worst method (BWM) technique was used to prioritize and determine the relative importance (weightage) of the risk factors. The weightages of the risk factors further used to rank the seven alternatives (HHD users) using Vlse Kriterijumska Optimizacija Kompromisno Resenje (VIKOR) technique. The outcomes of the BWM investigation showed that the three most significant risk factors responsible for MSDs are duration of working, poor working posture and un-ergonomic design. The outcome of the VIKOR technique exhibited that computer professionals were at the highest risk among all users. The risk factor priority must be used for designing a working strategy for the WFH situation which will help to mitigate the risks of MSDs. Keywords"

"Relevant literature This section is classified into two parts: (1) MSDs among HHD users and (2) soft computing tools used for decision-making. MSDs among HHD users The use of HHDs has become vital in the various work environments. Several epidemiology studies demonstrate that MSDs are prevalent among the users working with HHDs (Chiang and Liu 2016 ; Woo et al. 2016 ; Taib et al. 2016 ; Xie et al. 2017 ; Soria-Oliver et al. 2019 ). Significant associations of MSDs with physical (Chiang and Liu 2016 ; Woo et al. 2016 ; Xie et al. 2017 ) and psychosocial (Janwantanakul et al. 2012 ; Taib et al. 2016 ; Soria-Oliver et al. 2019 ) factors have been found in previous studies. Previous studies also observed that work-related factor (PF or PSF) is not a single factor that can develop MSDs. IFs such as gender, age, obesity, and smoking behaviour are also significant reason of MSDs development (Taib et al. 2016 ; Xie et al. 2017 ). Depending upon the severity of the pain, either MSDs can be at the initial level, or it converts into disability when not appropriately diagnosed. MSD at initial level is curable without difficulty, and it takes a month or less time to recover for a suffering person (Laisné et al. 2012 ; Kuijer et al. 2012 ). However, the treatment of disability is somewhat complicated, and it can take a long time for the individual to improve. Investigators have identified many factors that could account for a change from acute to chronic MSDs (Keefe et al. 2018 ). Identifying the responsible risk factors for the development of MSDs can assist to identify the risk level which will further help in deciding the preventive measures. Primary prevention helps in reducing the risk of the initial onset of a problem (Waongenngarm et al. 2018 ; Williams et al. 2018 ). To reduce the MSDs among HHD users, it is vital to think about the priority and relative importance of the risk factors. Previous researches reported in the literature have used various soft computing tools for identifying the risk level by appropriate decision-making strategies among various work environments. Soft computing tools used for decision-making In the reported literature, various soft computing techniques or approaches have been used by previous researchers to identify the risk level among various work environments. Castillo and Melin ( 2020 ) proposed a hybrid intelligent approach for forecasting the future trends of pandemic situations based on the COVID-19 time series of confirmed cases and deaths. Dansana et al. ( 2020 ) used deep learning algorithms to map the computed tomography and X-rays reports of COVID-19 patients for providing better and faster treatment. Melin et al. ( 2020a ) done a spatial evolution of different country maps for exploring COVID-19 pandemic situations by using an unsupervised neural network. Melin et al. ( 2020b ) implemented the concepts of neural network and fuzzy logic for predicting the COVID-19 time series in Mexico. These evolutionary approaches provided the efficient predictions of collected data for larger sample sizes. However, the pairwise comparison of multiple factors rating data was done previously by MCDM approaches mostly. Maldonado-Macías et al. ( 2014 ) evaluated the ergonomic compatibility of advanced technology used in manufacturing industries. Chiu and Hsieh ( 2016 ) applied fuzzy TOPSIS for improving the maintenance tasks in the aviation industry. Ahmadi et al. ( 2017 ) developed a scoring model for estimating the ergonomic risks to determine risky situations by using a mixture of MCDM approaches. Khandan et al. ( 2017 ) used fuzzy TOPSIS for evaluating the occupational disorders risk and ergonomic problems in workplaces. Khan et al. ( 2019 ) used the BWM method for prioritizing the risk factors of lower back pain in the industrial workers and advised that there is a need of MCDM techniques usage for solving the health problems faced by workers in various environments. There are various MCDM approaches used for evaluation of multiple criteria (risk categories) like AHP, ANP, DEMATEL, fuzzy TOPSIS, etc. (Ahmadi et al. 2017 ; Khandan et al. 2017 ). However, BWM was an efficient approach due to the merits of this approach as compared to other approaches with smaller amount comparisons of rating data from the experts and higher consistency in results (Rezaei 2015 , 2016 ; Khan et al. 2019 ). Also, VIKOR was used for ranking of alternatives because of its capability to precisely optimize the multiple factors using co-operation precedence methodology (Mohanty and Mahapatra 2014 ; Mohanty et al. 2018 ). Despite these strengths, the MCDM approaches suffer from certain restrictions also; both BWM and VIKOR techniques governed by the decision-maker choices, therefore, it is essential to select decision-maker wisely depending on the expertise in relevant areas. Most of the earlier investigations dedicated for finding risk factors of MSDs among various occupational groups. The longer duration usage of HHDs in awkward posture causes discomfort in HHD users. However, currently most of the office users and students use HHDs (user friendly) for extensive times than other type of technology devices used in the past due to COVID-19 pandemic. Till date, there is no such research available which used MCDM approach for the evaluation of priority and relative importance of MSD risk factors among HHD users. This research gap is filled by using the integrated approach (BWM and VIKOR techniques) for identifying the risk level of MSDs among HHD users. Risk factors of MSDs Previous studies (Janwantanakul et al. 2012 ; Xie et al. 2017 ) have described three categories of risk factors for MSDs, i.e. PF, PSF, and IF, and some subfactors among each category. The different categories of MSD risk factors among the HHD users have not been explained in detail. Therefore, firstly relevant literature related to different type of HHD users was (Chiang and Liu 2016 ; Woo et al. 2016 ; Taib et al. 2016 ; Xie et al. 2017 ; Soria-Oliver et al. 2019 ) explored and discovered the prevalent MSD risk factors among the HHD users. Figure 1 depicts a listing of the various categories of MSD risk factors (primary factors and subfactors). This listing was also evaluated by the expert team which includes four decision-makers and two experts from the institute project committee before finalization. Implementation of proposed integrated approach The current research was carried out by taking the help of four decision-makers with research specialization in Human Factors and Ergonomics, occupational health and safety. The instructions and purpose of the research were briefed to all decision-makers before taking their responses. The decision-makers gave their informed consents and provided the requested data. On the basis of the input/data provided by the decision-makers, the further processing was done as per the steps of BWM and VIKOR techniques. Risk priority determination using BWM technique The probable factors/subfactors of MSD risks listing given in Fig. 1 were ranked using BWM technique. This technique was used for weight calculation of the primary factors, i.e. work-related individual factors (IF), psychosocial factors (PSF), and physical factors (PF). Similarly, the calculations for relative and global weights of the all subfactors of risks were also done and ranked the factors/subfactors on the basis of obtained weights. Relative weight calculation/risk priority for primary risk factors In the current research, decision-makers rated the PF as the best risk factor among other risk factors and provided ratings of other risk factors compared to the best risk factor using a scale of 1 to 9. A similar process was also carried out for providing the ratings to worst risk factor (IF). Equations ( 1 ) and ( 2 ) helped to generate the BFO and OTW vectors for the pairwise evaluation among the best and worst risk factor. The pairwise evaluation matrix is given in Table 2 . The optimum weights ( ) and consistency coefficient ( ) of primary risk factors were computed using Eqs. ( 3 ) and ( 4 ). The value of is very close to zero, which shows higher reliability in the evaluations and outcomes. Relative weights calculation/ priority of the subfactors of risks The relative weights of the subfactors of risks were computed using Eqs. ( 1 ) and ( 2 ). The pairwise evaluation matrix for subfactor comparisons is presented in Table 3 . The relative ranking of the primary risk factors and ranks of the subfactors of risks are presented in Table 4 . It shows that out of three primary risk factors, PF is the best risk factor pursued by PSF, and IF, i.e. PF > PSF > IF. Additionally, Table 4 also exposes that among all subfactors of IF, the order of priority is as PA > AG > OB > SM > GE. Similarly, among subfactors of PSF, the order of priority is as JS > RW > JSA > TAT. Lastly, the order of priority is as DW > PO > PD > FE among the subfactors of PF. Table 4 also exhibits that duration of working, posture and poor design are the topmost three MSD risk subfactors, since their rank is top three among all other risk subfactors whereas smoking, task/activity type and obesity are the last three risk subfactors. The outcomes of our research are comparable to the prior investigations (Janwantanakul et al. 2012 ; Moom et al. 2015 ; Kaliniene et al. 2016 ; Abaraogu et al. 2018 ; Sasikumar and Binoosh 2020 ), who reported that heavy duration of working, posture, poor design, and job strain are the subfactors of risks that causes MSD among the HHD users. Evaluating alternatives using VIKOR After acquiring optimum weights of risk subfactors, the alternatives are ranked based on weights of subfactors using VIKOR technique. By using linguistic scale provided in Table 1 , the decision-makers were rated the all HHD alternatives with respect to the subfactor of risks. The rating given by the decision-makers is presented in “ Appendix ”. These average ratings from decision-maker were computed for each alternatives using Eq. ( 5 ). The average decision matrix is revealed in Table 5 . The maximum and minimum values of risk factors were computed using Eqs. ( 6 ) and ( 7 ). Equations ( 8 )–( 10 ) were used for computing the values of Sa, Ra and Qa (Table 6 ). The computer professional ( A 2) attains first rank, as it has lowermost Qa value and also fulfils both situations ( and attains first rank according to both R a and S a values as presented in Table 6 ). The obtained results of alternative ranking are in line with previous researches of numerous occupational groups (Widanarko et al. 2011 , 2013 ; Silva et al. 2016 ) and computer professionals, causes higher MSDs due to long duration of working."

"Appendix: The ratings for seven alternatives by four decision-makers See Table 7 . Abbreviations AG Best Best–worst method Best from others Coronavirus Duration of work Force exertion Gender Handheld device Individual factor Job satisfaction Job strain Multi-criteria decision-making Musculoskeletal disorders Obesity Others to worst Physical activity Physical factor Poor design Posture Psychosocial factor Repetitive work Smoking Task/activity type Vlse Kriterijumska Optimizacija Kompromisno Resenje Work-from-home Worst Criteria Optimum weights Weightage of each factors from the BWM technique Total number of risk factors or alternatives Function values for processing data Positive best outcome Negative best outcome Weighted and normalized Manhattan distance or Utility measure Majority rule utility and regret measures Opponent rule utility and regret measures Weighted and normalized Chebyshev distance or Regret measure Closeness coefficient or VIKOR index Consistency ratio Acknowledgements The authors would like to acknowledge the National Project Implementing Unit and the Ministry of Human Resources Development for funding support in this research. Funding This research was supported by Collaborative Research Scheme implemented by the National Project Implementing Unit (NPIU) funded by the Ministry of Human Resources Development, Government of India (No. 1-5727963012). Compliance with ethical standards Conflict of interest All the authors declare that they have no conflict of interests. Ethical approval All procedures performed in studies involving human participants as a decision-maker were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the research."

"NO-CC CODE"

"no"

"2023-06-06 23:35:14"

"Soft comput. 2023 Feb 3; 27(6):3283-3293"

"oa_package/74/7d/PMC7856850.tar.gz"

"PMC7906623"

"33636145"

"Introduction\nPatients admitted to hospital with COVID-19 show various clinical signs and symptoms (...TRUNCATED)

"Methods\nDatabases\nIn this study, we used data from two cohorts: the COVID-19@Spain cohort, a retr(...TRUNCATED)

"Results\nThe features of the patients in the cohorts used for this study were previously reported i(...TRUNCATED)

"Discussion\nWe identified three phenotypes based on demographics, underlying conditions, clinical a(...TRUNCATED)

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"Members of the REIPI-SEIMC COVID-19 group and COVID@HULP group are listed in appendix pp 23–29\nB(...TRUNCATED)

"Data sharing\nData collected for the study, including deidentified participant data and a data dict(...TRUNCATED)

"Supplementary Material\n \nAcknowledgments\nThe study was funded by Instituto de Salud Carlos III(...TRUNCATED)

"NO-CC CODE"

"no"

"2023-06-06 23:35:22"

"Lancet Infect Dis. 2021 Jun 23; 21(6):783-792"

"oa_package/8e/1b/PMC7906623.tar.gz"

"PMC7929434"

"33567449"

""

""

""

""

""

""

"See corresponding article on pages 924 and 984 .\nSee corresponding articles on pages XXX-XXX an(...TRUNCATED)

"The author reports no conflicts of interest."

"NO-CC CODE"

"no"

"2023-06-06 23:35:02"

"Am J Clin Nutr. 2021 Apr 31; 113(4):763-764"

"oa_package/87/2e/PMC7929434.tar.gz"

"PMC8079233"

"33935377"

"Introduction\nWith the advent of the COVID-19 pandemic, a massive amount of multimedia healthcare d(...TRUNCATED)

"Methodology\nThe process of medical image-based COVID-19 detection CNN-based classification model i(...TRUNCATED)

"Results and discussion\nIn this section, we present the multi-classification results followed by a (...TRUNCATED)

"Results and discussion\nIn this section, we present the multi-classification results followed by a (...TRUNCATED)

"Conclusion and future scope\nThe COVID-19 pandemic has clearly put a threat to human existence. Eff(...TRUNCATED)

"The demand for automatic detection of Novel Coronavirus or COVID-19 is increasing across the globe.(...TRUNCATED)

"Literature of review\nRecent developments in deep learning have been seen over the years in many fi(...TRUNCATED)

"Acknowledgements\nNo Applicable.\nFunding\nThis work was supported by the Deanship of Scientific Re(...TRUNCATED)

"NO-CC CODE"

"no"

"2023-06-06 23:35:29"

"Multimed Syst. 2023 Apr 28; 29(3):1729-1738"

"oa_package/63/8a/PMC8079233.tar.gz"