BackgroundInfluenza A viruses are a major cause of viral respiratory infections in humans, leading to a spectrum of diseases ranging from mild to severe, particularly in high-risk groups. Monitoring the frequency and evolution of A (H1N1)pdm09 viruses is essential for guiding vaccine strain selection and maintaining vaccine effectiveness.ObjectivesTo characterize the evolution of circulating influenza A (H1N1)pdm09 strains in Mar del Plata(MDQ), Argentina, and estimate vaccine efficacy from 2015 to 2020, before the SARS-CoV-2 pandemic.MethodsWe analyzed 46 influenza A (H1N1)pdm09 strains detected by RT-PCR from patients with acute respiratory infections in MDQ between 2015 and 2020. Phylogenetic analysis was performed using maximum likelihood, and vaccine efficacy was estimated with the Pepitope model.ResultsSeven genetic clades were identified: 6B in 2015, 6B.1 in 2016-2017, 6B.1A, 6B.1A.1, and 6B.1A.3 in 2018, 6B.1A.5A in 2019-2020, and 6B.1A.5a.2 in 2020. Genetic diversity and regional clustering suggested multiple strain introductions from other Argentinian regions or countries. The predicted vaccine efficacy was highest when the frequency of influenza A (H1N1)pmd09 was below 2%, decreasing as viral frequency increased, due to mutations occurring particularly in haemagglutinin epitopes B, C, and E.ConclusionOur findings highlight the dynamic evolution of influenza A (H1N1)pdm09 in MDQ and emphasize the importance of continuous molecular surveillance and regular vaccine updates. Additionally, the Pepitope model proved useful in estimating vaccine efficacy. The impact of vaccination in reducing viral frequency when circulating and vaccine strains are well-matched further supports annual influenza vaccination recommendations to minimize viral burden in the community.

Influenza viruses are one of the main agents causing acute respiratory infections (ARI) in humans resulting in a large amount of illness and death globally. The influenza viruses classification is based on the nomenclature proposed by the World Health Organization (WHO) that is widely accepted and used by the medical and scientific communities throughout the world. Since the pandemic in 2009, two subtypes of human influenza A viruses, A(H1N1)pdm09 and A(H3N2), and two lineages of influenza B, B/Victoria and B/Yamagata, have been responsible for the vast majority of cases each year. Within each subtype and lineage, different clades and genetic groups were described to reflect the continuous viral evolution, driven by antigenic drift. The WHO Global Influenza Surveillance and Response System (GISRS) studies human influenza viruses from >110 countries, to monitor circulating strains, understand epidemiology and evolution, and contribute to verify the vaccine effectiveness and update its formulation each year. A growing number of laboratories and research centers is contributing to this initiative by sequencing the whole viral genome or the hemagglutinin (HA) gene from local strains. Influenza clade classification is usually performed by phylogenetic analysis of HA gene sequences from circulating strains along with reference sequences, which is a time-consuming process and requires specific training and equipment. Alternatively, this can be done by comparing amino acid substitutions, either manually or by using inhouse scripts. While there are currently specific tools available for influenza classification, they have several limitations such as: (a) they require an alignment of the input data against reference sequences (which can be computationally expensive), (b) requirement of multiple ad hoc programs installed, (c) users should be familiar with the command line, (d) users must create a template containing cladedefining amino acid pattern by position, (e) only classifies sequences into type A or B and subtype/lineage but cannot discern clades or genetic groups, and (f) take into account only the most prevalent and recent influenza clades. Advanced machine learning techniques have proven to make accurate predictions, using algorithms that reveal patterns in large datasets. In the analysis of viral data, machine learning methods have been recently implemented, for example, in: COVIDEX, a tool that classifies complete genome nucleotide sequences of SARS-CoV-2 into lineages, a recent application for avian influenza clade classification, the prediction of phenotypes for human influenza A from proteomic input, and detection of new variants using ensemble learning. In this sense, we developed INFINITy, a tool based on alignmentfree machine learning for human influenza virus classification into subtypes and clades. INFINITy is a web application that runs on an internet connection without any installation and has a user-friendly interface. It is fast, sensitive, specific, and ready to implement. Additionally, it is available to run locally for R and Rstudio users as an R package. Furthermore, two docker images are available to secure the reproducibility of the results. INFINITy includes two classification models: one for complete HA sequences (FULL HA, for whole gene sequence length of 1700 bp) and other for the HA1 subunit coding sequence (HA1, for the initial 1030 bp of the HA gene). The influenza classification comprises 75 clades or genetic groups: 25 for A(H1N1)pdm09, 32 for A(H3N2), and 14 for B/Victoria and 4 for B/Yamagata (supporting information Table S1). The overall classification algorithm is divided into three phases:

Objective: To report a conjunctivitis outbreak in a neonatology intensive care unit (NICU) and determine the associated economic impact. Design: Prospective observational study. Setting: Centro de Educacion Medica e Investigaciones Clinicas (CEMIC) University Hospital, a private, tertiary-care healthcare institution in Buenos Aires, Argentina. Participants: The study included 52 NICU neonates and 59 NICU-related healthcare workers (HCWs) from CEMIC hospital. Methods: Neonates and HCWs were swabbed for real-time polymerase chain reaction (PCR) testing, viral culture, and typing by sequencing. Infection control measures, structural and logistic changes were implemented. Billing records were analyzed to determine costs. Results: From January 30 to April 28, 2018, 52 neonates were hospitalized in the NICU. Among them, 14 of 52 (21%) had bilateral conjunctivitis with pseudomembranes. Symptomatic neonates and HCWs were HAdV-D8 positive. Ophthalmological symptoms had a median duration of 18 days (IQR, 13-24.5). PCR positivity and infectious range had a median duration of 18.5 days. As part of containment measures, the NICU and the high-risk pregnancy unit were closed to new patients. The NICU was divided into 2 areas for symptomatic and asymptomatic patients; a new room was assigned for the general nursery, and all deliveries from the high-risk pregnancy unit were redirected to other hospitals. The outbreak cost the hospital US$205,000: implementation of a new nursery room and extra salaries cost US$30,350 and estimated productivity loss during 1 month cost US$175,000. Conclusions: Laboratory diagnosis confirmed the cause of this outbreak as HAdV-D8. The immediate adoption and reinforcement of rigorous infection control measures limited the nosocomial viral spread. This outbreak represented a serious institutional problem, causing morbidity, significant economic loss, and absenteeism.

Human adenoviruses (HAdV) are one of the most frequent causes of respiratory infections around the world, causing mild to severe disease. In Argentina, many studies focused on the association of HAdV respiratory infection with severe disease and fatal outcomes leading to the discovery in 1984 of a genomic variant 7h associated with high fatality. Although several molecular studies reported the presence of at least 4 HAdV species (B, C, D and E) in Argentina, few sequences were available in the databases. In this study, sequences from the hexon gene region were obtained from 141 patients as a first approach to assess the genetic diversity of HAdVs circulating in Buenos Aires, Argentina. Phylogenetic analysis of these sequences and others recovered from public databases confirmed the circulation of the four above-mentioned species represented by 11 genotypes, with predominance in species B and C and shifts in their proportion in the studied period (2000 to 2018). The variants detected in Argentina, for most of the genotypes, were similar to those already described in other countries. However, uncommon lineages belonging to genotypes C2, C5 and E4 were detected, which might indicate the circulation of local variants and will deserve further studies of whole-genome sequences.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Lower acute respiratory infections (ARI) are a frequent cause of morbidity and mortality in infants, respiratory viruses being the major causative agents. The aim of this work was to determine the respiratory pathogen frequency, the clinical characteristics and the outcome in infants <2 months old hospitalized with ARI. A retrospective study was performed during a five-year period (2008-2011, 2014-2016). Respiratory viruses and atypical bacteria were studied using the FilmArray-Respiratory Panel. Demographic and clinical characteristics, hospitalization course and outcomes were evaluated. Of the 137 infants <2 months old hospitalized with ARI studied, a 94.9% positivity rate as determined in 117 infants with community-acquired infection and 20.0% in 20 infants who acquired the infection during their birth hospitalization in the neonatal intensive care units (NICU) (nosocomial ARI) (p < 0.001). In infants with community-acquired infection, Respiratory syncytial virus (RSV) (52.1%) and Rhinovirus/Enterovirus (RV/EV) (41.0%) were the most frequent detected pathogens. Coinfections were determined in one quarter of the infants, RSV-RV/EV being the most frequent combination. In infants with nosocomial infection, RV/EV, RSV or Parainfluenza-3 were detected as single pathogens. Most infants with community-acquired infection presented lower ARI (81.2%) while most infants in the NICU had upper ARI (55.0%). The median length of stay (LOS) in infants with community-acquired ARI was 4 days (IQR: 2-6). Positive infants with nosocomial infection had longer median LOS (71 days[ IQR:42-99]) compared to negative infants (58 days [IQR: 49-71]) (p = 0.507). Respiratory viruses were detected as the major causative agents of community-acquired infection in hospitalized infants <2-months old, RSV and RV/EV being the most frequently detected. Although a low pathogen positivity rate was observed in infants with nosocomial infection, they may prolong the LOS. (C) 2020 Asociacion Argentina de Microbiologia. Published by Elsevier Espana, S.L.U.

Delineate risk factors associated with severe hypoxemia (O2 sat ≤87%) in infants and children younger than 2 years hospitalized with single pathogen HRV infection.

Rhinoviruses were detected as sole pathogens in 6 preterm infants who developed severe respiratory infections while hospitalized in a neonatal intensive care unit. We confirmed 2 nosocomial rhinovirus transmission episodes and describe the genetic diversity of rhinovirus strains that circulated simultaneously during a winter season.

Background: Acute respiratory infections (ARI) are a leading cause of morbidity and mortality worldwide. There is a need to demonstrate the clinical impact of using the new, rapid and sensitive molecular assays in prospectively designed studies. Objectives: To study the impact on medical management of a rapid molecular assay in patients with respiratory infections. Study design: A prospective, randomized, non-blinded study was performed in patients presenting to the Emergency Department during two respiratory seasons (2016-2017). Diagnosis was performed by FilmArray Respiratory Panel (FilmArray-RP) or by immunofluorescence assay (IFA). Results: A total of 432 patients (156 children and 276 adults) were analyzed. Diagnosis with FilmArray-RP was associated with significant changes in medical management including withholding antibiotic prescriptions (OR: 15.52, 95% CI: 1.99-120.83 in adults and OR: 12.23, 95% CI: 1.56-96.09 in children), and reduction in complementary studies in children (OR: 9.64, 95% CI: 2.13-43.63) compared to IFA. Decrease in oseltamivir prescriptions was significantly higher in adults in the FilmArray-RP group (p= 0.042; OR: 1.19, 95% CI: 0.512.79) compared to adults managed with IFA. Diagnostic yield was significantly higher by FilmArray-RP (81%) than by IFA (31%)(p < 0.001). The median time from sample collection to reporting was 1 h 52 min by FilmArray-RP and 26 h by IFA (p < 0.001). Conclusions: The high respiratory viruses' detection rate and availability of results within two hours when using FilmArray-RP were associated with decreases in antibiotic prescriptions and complementary studies and more accurate use of oseltamivir.

Background: Influenza A and B (Flu A/B), parainfluenza (PIV) and respiratory syncytial virus (RSV) cause lower and upper respiratory tract disease (LRD-URD) with significant clinical impact on patients with hematological malignancies (HM) or hematopoietic stem cell transplantation (HSCT). Rhinoviruses (HRV) are being increasingly detected in these infections, although their clinical impact remains a matter of debate. Our objective was to describe and compare clinical characteristics and outcomes of patients with HM and HSCT with LRD-URD caused by HRV versus non-HRV: Flu A/B, PIV and RSV. Methods & Materials: Prospective observational study. We compared HRV (G1) vs. Flu A/B, PIV and RSV (G2) respiratory infections in patients with HM and HSCT between January 2013 and September 2017. Chi-square analysis and Kruskal-Wallis test were used for categorical and continuous variables, respectively. Results: We enrolled 114 episodes: 45 in G1, 69 in G2. Both groups had patients with similar hematological diseases and stages, being lymphoma and acute leukemia the most frequent. Steroid therapy (20% vs 50.7%, p = 0.001) was significantly higher in G2, while use of biologic agents (40% vs 17.4%, p = 0.007), lymphopenia (33% vs 16.2% p = 0.034) and clinical presentation during preengraftment (20% vs 5.8%, p = 0.032) was higher in G1. Rhinorrhea was the most common symptom in G1 (71.1% vs 46.4%, p = 0.009). Other symptoms had similar frequencies in both groups. Over 50% of all infections presented as LRD (52% vs 50.7%, p = 0.968). Hypoxemia presented in similar rates (28.9% vs 27.5%, p = 0.875). The most common tomographic infiltrates were alveolar pattern and bilateral extension. In G1, co-pathogens in respiratory specimens were isolated in three patients (6.7% vs 0% p = 0.053) and other three had detectable plasma CMV viral load (6.7% vs 0% p = 0.012). Hospitalization was required in 58.8% of cases, with no significant difference between both groups. The 30-day overall mortality rate due to G1 and G2 infections were 6.7% and 7.2%, respectively (p = 1). Conclusion: Patients with HM or HSCT and HRV infections had similar clinical picture and outcome to common respiratory viruses, with significant morbidity. Therefore, active diagnostic approaches are required, especially in patients with lymphopenia or use of biologic agents.

Background We have previously estimated that respiratory syncytial virus (RSV) was associated with 22% of all episodes of (severe) acute lower respiratory infection (ALRI) resulting in 55 000 to 199 000 deaths in children younger than 5 years in 2005. In the past 5 years, major research activity on RSV has yielded substantial new data from developing countries. With a considerably expanded dataset from a large international collaboration, we aimed to estimate the global incidence, hospital admission rate, and mortality from RSV-ALRI episodes in young children in 2015.Methods We estimated the incidence and hospital admission rate of RSV-associated ALRI (RSV-ALRI) in children younger than 5 years stratified by age and World Bank income regions from a systematic review of studies published between Jan 1, 1995, and Dec 31, 2016, and unpublished data from 76 high quality population-based studies. We estimated the RSV-ALRI incidence for 132 developing countries using a risk factor-based model and 2015 population estimates. We estimated the in-hospital RSV-ALRI mortality by combining in-hospital case fatality ratios with hospital admission estimates from hospital-based (published and unpublished) studies. We also estimated overall RSV-ALRI mortality by identifying studies reporting monthly data for ALRI mortality in the community and RSV activity.Findings We estimated that globally in 2015, 33.1 million (uncertainty range [UR] 21.6-50.3) episodes of RSV-ALRI, resulted in about 3.2 million (2.7-3.8) hospital admissions, and 59 600 (48 000-74 500) in-hospital deaths in children younger than 5 years. In children younger than 6 months, 1.4 million (UR 1.2-1.7) hospital admissions, and 27 300 (UR 20 700-36 200) in-hospital deaths were due to RSV-ALRI. We also estimated that the overall RSV-ALRI mortality could be as high as 118 200 (UR 94 600-149 400). Incidence and mortality varied substantially from year to year in any given population.Interpretation Globally, RSV is a common cause of childhood ALRI and a major cause of hospital admissions in young children, resulting in a substantial burden on health-care services. About 45% of hospital admissions and inhospital deaths due to RSV-ALRI occur in children younger than 6 months. An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group.

Determining the incidence of influenza and other major viruses causing severe respiratory illness and the cost for hospitalization are important information for middle income countries like Argentina since such information will be useful for preventive measures like vaccine implementation and control measures. However there are several important major points the authors need to revise before considering for acceptance. Major comments Major Compulsory Revisions The authors conducted a prospective cohort study of approximately 1800 children equal or less than 5 years enrolled in CEMIC, a nonprofit health insurance plan. Social-economic status and background of families enrolling into this insurance program should be elaborated. There must be other insurance plans and people taking different insurance plan may have different social-economic background. The author mentioned that the children were entitled to assess two hospitals and emergency clinics for treatment. The names and/or location of the hospitals and the clinics will be important to understand which region in Argentina the study was conducted. Viral distribution may differ based on different geographical location, even in the same country. The authors should elaborate the geographic and social-economic background of the study population. Th author mentioned that a convenience sample of children presenting to an emergency room with acute respiratory sign or symptom were enrolled into the study only because of the insufficient resources for a random selection. Similarly, asymptomatic children were enrolled by convenience sampling. How many emergency clinics were assessable by the CEMIC insurance holders and which emergency clinic was selected for this study? Which hospital was selected for the asymptomatic children?

Las infecciones respiratorias agudas producen una importante morbimortalidad y comúnmente son causadas por virus. En Argentina, los programas de vigilancia epidemiológica se basan en la detección de antígenos virales por inmunofluorescencia (IF), aunque es bien conocido que los métodos moleculares son más sensibles. El panel respiratorio (PR) FilmArray (PR-FilmArray) es un equipo comercial automatizado de PCR múltiples que detecta 17 virus respiratorios y 3 bacterias, en un sistema cerrado que requiere 5min de procesamiento y una 1h de instrumentación. Se evaluó un total de 315 muestras respiratorias de niños menores de 6 años con infecciones respiratorias agudas por IF para 8 virus respiratorios y por RT-PCR para rinovirus. Posteriormente, estas muestras se estudiaron con el PR-FilmArray. La frecuencia de positividad al considerar los 9 virus estudiados por IF y RT-PCR fue del 75%; por PR-FilmArray fue del 92%. El porcentaje de acuerdo positivo entre ambas metodologías fue del 70,5% y el de acuerdo negativo fue del 99,6% (intervalo de confianza 95%: 65,5-75,1 y 99,2-99,8, respectivamente). El PR-FilmArray permitió obtener un mayor diagnóstico positivo (97%) y detectó otros virus, como los coronavirus NL63, 229E, OC43 y HKU1 (10%) y los bocavirus (18%). Además, permitió identificar coinfecciones múltiples (39%) con 2, 3, 4 y hasta 5 virus. Actualmente, la IF continúa siendo el método más utilizado en los países latinoamericanos para el diagnóstico de virus respiratorios por su bajo costo, por su capacidad para procesar un alto número de muestras simultáneamente y porque los resultados de los virus más frecuentes están disponibles en 5h. Sin embargo, la futura incorporación de métodos moleculares aumentaría notablemente la capacidad diagnóstica.

Although information about the incidence of viral respiratory illnesses and their associated cost can help health officials explore the value of interventions, data are limited from middle-income countries.

Fil: Carballal, Guadalupe. Centro de Educacion Medica e Investigaciones Clinicas Norberto Quirno. Direccion de Asistencia Medica. Departamento de Analisis Clinicos; Argentina

Background: Human rhinoviruses (HRV) are recognized as a cause of upper and lower acute respiratory infections (ARI). The circulating species and their clinical impact were not described in Argentina.Objectives: To describe the molecular epidemiology of HRV in children and to determine the association of HRV species with outcome and severity.Study design: Hospitalized and outpatients children <6 years old with ARI without comorbidities (n = 620) were enrolled (2008-2010). Demographic, clinical data and outcome were analyzed. HRV were identified by RT-PCR. Phylogenetic analysis and demographic reconstruction for HRV were performed in selected samples.Results: HRV were detected in 2521620(40.6%) of children; 8.5% in viral coinfection. Bronchiolitis (55%) and pneumonia (13%) were the most frequent clinical diagnosis. Of 202 inpatients with HRV: 72% required oxygen supplementation, 11% intensive care unit and 3% mechanical ventilation. HRV were identified as a risk factor for hospitalization (OR: 2.47).All three HRV species were detected being HRV-A (55%) and HRV-C (43%) the most frequent; HRV-B was infrequent (2%). Of 44 sequenced HRV, 30 genotypes were detected. Seven of them were the most prevalent and circulated during limited periods of time. The demographic reconstruction revealed a constant population size and a high turnover rate of genotypes. Demographic and clinical outcome were similar for HRV-A and HRV-C infections.Conclusion: This study highlights the clinical impact of HRV in children without comorbidities as a cause of lower ARI and hospitalization. The high frequency of HRV infections may be associated with the simultaneous circulation of genotypes and their high turnover rate. (C) 2014 Elsevier B.V. All rights reserved.

Palabras clave: rinovirus, virus respiratorios, RT-PCR tiempo real, infeccion respiratoria aguda, bronquiolitis, ninos Abstract Rhinoviruses. Frequency in nonhospitalized children with acute respiratory infection. Molecular methods for human rhinoviruses (HRV) have increased the sensitivity in their diagnosis. HRV may cause acute respiratory infections (ARI) of the upper and lower respiratory tract. HRV infection during childhood is a predictor of asthma development. In this study, the HRV frequency in outpatient children with ARI was de- termined, and their clinical features and previous conditions were evaluated. A total of 186 respiratory samples of children under 6 year old attending the CEMIC pediatric emergency room from June 1, 2008 to May 31, 2010, were studied. Classical respiratory viruses were detected by immunofluorescence. A real time RT-PCR that ampli - fies part of the 5' non coding genomic region was used for HRV detection. Viral detection was obtained in 61% of children. The frequency was: 27% for HRV, 16% for respiratory syncytial virus (RSV), 9% for influenza, 8% for parainfluenza, 7% for metapneumovirus and 0.5% for adenovirus. Dual coinfection was detected in 8 children and HRV were the most frequent, detected in 4 of them. HRV circulated during the two year period of the study, with peaks during winter and spring. No clinical difference was observed between patients with or without HRV, except an increase percent of children with HRV without fever. HRV were the most frequent viruses detected in this population, mainly in children under 2 year old, the second cause of bronchiolitis after RSV and more frequently detected in children exposed to passive smoking (OR = 2.91; p = 0.012), and were detected as the sole etiologic agent in 28% of bronchiolitis.