Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Rapid and on-site wireless immunoassay of respiratory virus aerosols via hydrogel-modulated resonators.

Rapid and accurate detection of respiratory virus aerosols is highlighted for virus surveillance and infection control. Here, we report a wireless immunoassay technology for fast (within 10 min), on-site (wireless and battery-free), and sensitive (limit of detection down to fg/L) detection of virus antigens in aerosols. The wireless immunoassay leverages the immuno-responsive hydrogel-modulated radio frequency resonant sensor to capture and amplify the recognition of virus antigen, and flexible readout network to transduce the immuno bindings into electrical signals. The wireless immunoassay achieves simultaneous detection of respiratory viruses such as severe acute respiratory syndrome coronavirus 2, influenza A H1N1 virus, and respiratory syncytial virus for community infection surveillance. Direct detection of unpretreated clinical samples further demonstrates high accuracy for diagnosis of respiratory virus infection. This work provides a sensitive and accurate immunoassay technology for on-site virus detection and disease diagnosis compatible with wearable integration.

Evaluating an extraction-free sample preparation method for multiplex detection of SARS-Cov-2, influenza A/B, and RSV with implementation on a microfluidic chip.

Following the relaxation of COVID-19 restrictions, other respiratory viruses such as influenza and respiratory syncytial virus (RSV), whose transmission were decreased due to COVID-19 precautions, are rising again. Because of similar clinical features and reported co-infections, multiplex detection of SARS-CoV-2, influenza A/B, and RSV is required to use specific treatments. This study assessed an extraction-free sample preparation (heat treatment at 95°C for 3 minutes) for multiplex detection using rRT-PCR. Despite an observed Ct-delay (∆Ct) averageing 1.26 compared to the standard method, an acceptable total sensitivity of 92 % and a negative predictive value (NPV) of 96 % were obtained. Moreover, Implementation on a microfluidic chip demonstrated efficiency, maintaining an excellent correlation (R2=0.983) with the standard method. Combining this extraction-free procedure with rRT-PCR on a microfluidic chip seems promising, because it simplifies the design and reduces the cost and complexity of the integrated assay for multiplex detection of SARS-CoV-2, influenza A/B, and RSV.

Peptide-Based electrochemical potential Scanning: A novel approach for disulfide manipulation in pediatric Respiratory syncytial virus detection.

Respiratory syncytial virus (RSV) poses a significant risk to children under two years old, necessitating rapid and accurate diagnostic methods. This study introduces an innovative approach using peptides and electrochemical potential scanning for RSV detection. By replacing enzymatic catalysis with electrochemical scanning, the method simplifies the process and reduces costs. Unbound peptides undergo potential-induced disulfide bridge opening, while target-bound peptides remain protected. After removing the target protein, copper ions and a reduced short peptide promote disulfide bridge formation, leading to crosslinking and passivation of the electrode surface. The degree of polymerization and passivation correlates with the target protein levels, generating a signal. This novel method offers enhanced sensitivity, specificity, and scalability, potentially revolutionizing RSV diagnostics in children under two years old. By addressing the limitations of traditional assays, it provides a cost-effective, rapid, and efficient approach for early RSV detection and improved clinical outcomes in this vulnerable population.

Respiratory syncytial virus surge in 2022 caused by lineages already present before the COVID-19 pandemic.

In 2022, Austria experienced a severe respiratory syncytial virus (RSV) epidemic with an earlier-than-usual start (Weeks 35/2021-45/2022) and increased numbers of pediatric patients in emergency departments. This surge came 2 years after a season with no cases detected as a result of coronavirus disease 2019 nonpharmaceutical interventions. We analyzed epidemiologic patterns and the phylodynamics of RSV based on approximately 30 800 respiratory specimens collected year-round over 10 years from ambulatory and hospitalized patients from 248 locations in Austria. Genomic surveillance and phylogenetic analysis of 186 RSV-A and 187 RSV-B partial glycoprotein sequences collected from 2018 to 2022 revealed that the 2022/2023 surge was driven by RSV-B in contrast to the surge in the 2021/2022 season that was driven by RSV-A. Whole-genome sequencing and phylodynamic analysis indicated that the RSV-B strain GB5.0.6a was the predominant genotype in the 2022/2023 season and emerged in late 2019. The results provide insight into RSV evolution and epidemiology that will be applicable to future monitoring efforts with the advent of novel vaccines and therapeutics.

Evaluation of the Alinity m Resp-4-Plex Assay for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2, Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus.

The rapid emergence of the coronavirus disease 2019 (COVID-19) pandemic has introduced a new challenge in diagnosing and differentiating respiratory infections. Accurate diagnosis of respiratory infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is complicated by overlapping symptomology, and stepwise approaches to testing for each infection would lead to increased reagent usage and cost, as well as delays in clinical interventions. To avoid these issues, multiplex molecular assays have been developed to differentiate between respiratory viruses in a single test to meet clinical diagnostic needs. To evaluate the analytical performance of the FDA emergency use authorization (EUA)-approved Abbott Alinity m resp-4-plex assay (Alinity m) in testing for SARS-CoV-2, influenza A virus, influenza B virus, and respiratory syncytial virus (RSV), we compared its performance to those of both the EUA-approved Cepheid Xpert Xpress SARS-CoV-2, influenza A/B virus, and RSV assay (Xpert Xpress) and the EUA-approved Roche Cobas SARS-CoV-2 and influenza A/B virus assay (Cobas) in a single-center retrospective analysis. High concordance was observed among all three assays, with kappa statistics showing an almost perfect agreement (>0.90). The limit of detection (LOD) results for SARS-CoV-2 showed the Alinity m exhibiting the lowest LOD at 26 copies/mL, followed by the Cobas at 58 copies/mL and the Xpert Xpress at 83 copies/mL, with LOD results for the influenza A virus, influenza B virus, and RSV viral targets also showing equivalent or better performance on the Alinity m compared to the other two platforms. The Alinity m can be used as a high-volume testing platform for SARS-CoV-2, influenza A virus, influenza B virus, and RSV and exhibits analytical performance comparable to those of both the Xpert Xpress and Cobas assays. IMPORTANCE The rapid emergence of SARS-CoV-2 has introduced a new challenge in diagnosing and differentiating respiratory infections, especially considering the overlapping symptomology of many of these infections and differences in clinical interventions depending on the pathogen identified. To avoid these issues, multiplex molecular assays like the one described in this article need to be developed to differentiate between the most common respiratory pathogens in a single test and most effectively meet clinical diagnostic needs.

Multicenter study evaluating one multiplex RT-PCR assay to detect SARS-CoV-2, influenza A/B, and respiratory syncytia virus using the LabTurbo AIO open platform: epidemiological features, automated sample-to-result, and high-throughput testing.

Since the Coronavirus 19 (COVID-19) pandemic, several SARS-CoV-2 variants of concern (SARS-CoV-2 VOC) have been reported. The B.1.1.7 variant has been associated with increased mortality and transmission risk. Furthermore, cluster and possible co-infection cases could occur in the next influenza season or COVID-19 pandemic wave, warranting efficient diagnosis and treatment decision making. Here, we aimed to detect SARS-CoV-2 and other common respiratory viruses using multiplex RT-PCR developed on the LabTurbo AIO 48 open system. We performed a multicenter study to evaluate the performance and analytical sensitivity of the LabTurbo AIO 48 system for SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) using 652 nasopharyngeal swab clinical samples from patients. The LabTurbo AIO 48 system demonstrated a sensitivity of 9.4 copies/per PCR for N2 of SARS-CoV-2; 24 copies/per PCR for M of influenza A and B; and 24 copies/per PCR for N of RSV. The assay presented consistent performance in the multicenter study. The multiplex RT-PCR applied on the LabTurbo AIO 48 open platform provided highly sensitive, robust, and accurate results and enabled high-throughput detection of B.1.1.7, influenza A/B, and RSV with short turnaround times. Therefore, this automated molecular diagnostic assay could enable streamlined testing if COVID-19 becomes a seasonal disease.

Comparison of 11 respiratory pathogens among hospitalized children before and during the COVID-19 epidemic in Shenzhen, China.

BACKGROUND: The effect of SARS-CoV-2 on existing respiratory pathogens in circulation remains uncertain. This study aimed to assess the impact of SARS-CoV-2 on the prevalence of respiratory pathogens among hospitalized children. METHODS: This study enrolled hospitalized children with acute respiratory infections in Shenzhen Children's Hospital from September to December 2019 (before the COVID-19 epidemic) and those from September to December 2020 (during the COVID-19 epidemic). Nasopharyngeal swabs were collected, and respiratory pathogens were detected using multiplex PCR. The absolute case number and detection rates of 11 pathogens were collected and analyzed. RESULTS: A total of 5696 children with respiratory tract infection received multiplex PCR examination for respiratory pathogens: 2298 from September to December 2019 and 3398 from September to December 2020. At least one pathogen was detected in 1850 (80.5%) patients in 2019, and in 2380 (70.0%) patients in 2020; the detection rate in 2020 was significantly lower than that in 2019.The Influenza A (InfA) detection rate was 5.6% in 2019, but 0% in 2020. The detection rates of Mycoplasma pneumoniae, Human adenovirus, and Human rhinovirus also decreased from 20% (460), 8.9% (206), and 41.8% (961) in 2019 to 1.0% (37), 2.1% (77), and 25.6% (873) in 2020, respectively. In contrast, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased from 6.6% (153), 9.9% (229), and 0.5% (12) in 2019 to 25.6% (873), 15.5% (530), and 7.2% (247) in 2020, respectively (p < 0.0001). CONCLUSIONS: Successful containment of seasonal influenza as a result of COVID-19 control measures will ensure we are better equipped to deal with future outbreaks of both influenza and COVID-19.Caused by virus competition, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased in Shenzhen,that reminds us we need to take further monitoring and preventive measures in the next epidemic season.

Molecular Mechanism of Jinchan Oral Liquid in the Treatment of Children with Respiratory Syncytial Virus Pneumonia Based on Network Pharmacology and Molecular Docking Technology.

OBJECTIVE: Exploration of the underlying molecular mechanism of Jinchan Oral Liquid (JOL) in treating children with the respiratory syncytial virus (RSV) pneumonia to provide new evidence for the clinical application. METHODS: The active components and target genes of JOL were screened by the TCMSP database. The targets of RSV pneumonia were obtained from the GeneCards, OMIM, DrugBank, and PharmGKB database. Then, we constructed the active component-target network and screened the core genes. The overlaps were screened for PPI network analysis, GO analysis, and KEGG analysis. Finally, result validation was performed by molecular docking. RESULTS: According to the screening criteria of the ADME, 74 active compounds of JOL were obtained; after removing redundant targets, we selected 180 potential targets. By screening the online database, 893 RSV pneumonia-related targets were obtained. A total of 82 overlapping genes were chosen by looking for the intersection. The STRING online database was used to acquire PPI relationships, and 16 core genes were obtained. GO and KEGG analyses showed that the main pathways of JOL in treating RSV pneumonia include TNF signaling pathway and IL17 signaling pathway. The molecular docking results showed that the active compounds of JOL had a good affinity with the core genes. CONCLUSION: In this study, we preliminarily discussed the main active ingredients, related targets, and pathways of JOL and predicted the pharmacodynamic basis and the potential therapeutic mechanisms of RSV pneumonia. In summary, the network pharmacology strategy may be helpful for the discovery of multitarget drugs against complex diseases.

Comparison of three air samplers for the collection of four nebulized respiratory viruses - Collection of respiratory viruses from air.

Viral respiratory tract infections are a leading cause of morbidity and mortality worldwide. Unfortunately, the transmission routes and shedding kinetics of respiratory viruses remain poorly understood. Air sampling techniques to quantify infectious viruses in the air are indispensable to improve intervention strategies to control and prevent spreading of respiratory viruses. Here, the collection of infectious virus with the six-stage Andersen cascade impactor was optimized with semi-solid gelatin as collection surface. Subsequently, the collection efficiency of the cascade impactor, the SKC BioSampler, and an in-house developed electrostatic precipitator was compared. In an in vitro set-up, influenza A virus, human metapneumovirus, parainfluenza virus type 3, and respiratory syncytial virus were nebulized and the amount of collected infectious virus and viral RNA was quantified with each air sampler. Whereas only low amounts of virus were collected using the electrostatic precipitator, high amounts were collected with the BioSampler and cascade impactor. The BioSampler allowed straight-forward sampling in liquid medium, whereas the more laborious cascade impactor allowed size fractionation of virus-containing particles. Depending on the research question, either the BioSampler or the cascade impactor can be applied in laboratory and field settings, such as hospitals to gain more insight into the transmission routes of respiratory viruses.

Prognosis of hospitalized children under 2 years of age with co-detection of influenza A and respiratory syncytial virus at the healthcare facility / Prognóstico de crianças hospitalizadas menores de 2 anos com codetecção de influenza A e vírus sincicial respiratório no local de atendimento

Abstract Objectives: the aim of this study is to evaluate the impact of co-detection of Flu A and RSV using rapid immunochromatographic tests at the point of care, in pediatric patients under 2 years of age in a general hospital. Methods: a retrospective cohort study was conducted to analyze clinical outcomes in hospitalized infants with viral respiratory disease with positive results of rapid immunochromatographic test for RSV and/or Flu-A, from 2013 to 2018. A logistic regression model was adjusted to analyze predictors of orotracheal intubation during hospitalization. Results: we analyzed 220 cases: RSV (192), Flu-A (9), co-detection (19). Lethality rate was 1.8% (2 cases), and 88% (194) were under 1 year of age. Mean time of hospitalizations was higher in patients with co-detection. Variables significantly associated with orotracheal intubation were: younger age in months, comorbidities, RSV and Flu-A co-detection, and bacterial pneumonia during hospitalization. Conclusions: RSV and Flu-Aco-detection was associated with the least favorable clinical prognoses in this study. Rapid test diagnosis may provide important information at the point of care, because molecular panels are not widely accessible in general hospitals. Rapid diagnosis allows timely evaluation and treatment.

Resumo Objetivos: avaliar o impacto da codetecção de Influenza A (FluA) e Vírus Sincicial Respiratório (VSR) por meio de testes imunocromatográficos rápidos em tempo real, em pacientes menores de 2 anos em hospital público e universitário. Métodos: estudo de coorte retrospectivo foi conduzido para analisar os desfechos clínicos de crianças hospitalizadas com doença respiratória viral com resultados positivos do teste rápido imunocromatográfico para VSR e/ou FluA, de 2013 a 2018. Um modelo de regressão logística foi ajustado para analisar preditores de intubação orotraqueal durante a internação. Resultados: foram analisados 220 casos: RSV (192), FluA (9) eco-detecção (19). A letalidade foi de 1,8% (2 casos) e 88% (194) casos em menores de 1 ano. O tempo médio de internação foi maior nos pacientes com codetecção. As variáveis significativamente associadas à intubação orotraqueal foram: menor idade em meses, comorbidades, codetecção de VSR e Flu-A e pneumonia bacteriana durante a internação. Conclusões: codetecção VSR e FluA foi associada a prognósticos clínicos desfavoráveis. O teste rápido fornece informações importantes a beira-leito, pois os painéis moleculares não são amplamente acessíveis em hospitais públicos. O diagnóstico rápido permite a avaliação e tratamento oportunos.

Epidemiology and factors associated with the severity of viral acute lower respiratory infection in children hospitalized in Manaus, Amazonas, in 2017-2018: An observational study.

ABSTRACT: To investigate the epidemiology and factors associated with the severity of viral acute lower respiratory infection (ALRI) in children hospitalized in Manaus, Amazonas, in 2017 to 2018.Retrospective cohort study of children hospitalized at the Hospital and Emergency Room Delphina Rinaldi Abdel Aziz, in Manaus, from April 01, 2017 to August 31, 2018, with a clinical diagnosis of ALRI and nasopharyngeal aspirates positive for at least 1 respiratory virus.One hundred forty-six children aged 0.2 to 66 months (median 7 months) were included. Patients were divided into 2 groups according to the disease severity classified by an adapted Walsh et al score: moderate disease, score 0-4, n = 66 (45.2%) and severe disease, score 5-7, n = 80 (54.8%). A greater number of viral ALRI cases were observed in the rainiest months. Respiratory syncytial virus was the most prevalent (n = 103, 70.3%), followed by metapneumovirus (n = 24, 16.4%), influenza virus (n = 17, 11.6%), parainfluenza virus (n = 11, 7.5%), and adenovirus (n = 4, 2.7%). Co-detections of 2 to 3 viruses were found in 12 (8.2%) patients. The presence of viral coinfection was an independent risk factor for disease severity (adjusted relative risk [RR] 1.53; 95% CI 1.10-2.14). Twelve patients (8.2%) died, all with severe disease. Risk factors for death were shock (adjusted RR 10.09; 95% CI 2.31-43.90) and need for vasoactive drugs (adjusted RR 10.63; 95% CI 2.44-46.31).There was a higher incidence of viral ALRI in Manaus in the rainy season. Respiratory syncytial virus was the most prevalent virus. The presence of viral coinfection was an independent risk factor for disease severity.

Absence of detection of RSV and influenza during the COVID-19 pandemic in a Brazilian cohort: Likely role of lower transmission in the community.

BACKGROUND: Respiratory syncytial virus (RSV) and influenza are prevalent seasonal community viruses. Although not completely understood, SARS-CoV-2 may have the same means of transmission. Preventive social measures aimed at preventing SARS-CoV-2 spread could impact transmission of other respiratory viruses as well. The aim of this study is to report the detection of RSV and influenza during the period of social distancing due to COVID-19 pandemic in a heavily affected community. METHODS: Prospective study with pediatric and adult populations seeking care for COVID-19-like symptoms during the fall and winter of 2020 at two hospitals in Southern Brazil. RT-PCR tests for SARS-CoV-2, influenza A (Flu A), influenza B (Flu B) and respiratory syncytial virus (RSV) was performed for all participants. RESULTS: 1435 suspected COVID-19 participants (1137 adults, and 298 children). were included between May and August. Median age was 37.7 years (IQR = 29.6-47.7), and 4.92 years (IQR = 1.96-9.53), for the adult and child cohorts, respectively. SARS-CoV-2 was positive in 469 (32.7%) while influenza and RSV were not detected at all. CONCLUSIONS: Measures to reduce SARS-CoV-2 transmission likely exerted a huge impact in the spread of alternate respiratory pathogens. These findings contribute to the knowledge about the dynamics of virus spread. Further, it may be considered for guiding therapeutic choices for these other viruses.

Airflow dispersion during common neonatal resuscitation procedures: A simulation study.

BACKGROUND: Aerosol generating medical procedures (AGMPs) are common during newborn resuscitation. Neonates with respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may pose a risk to healthcare workers. International guidelines differ on methods to minimize the risk due to limited data. OBJECTIVE: We examined the expiratory airflow dispersion during common neonatal resuscitation AGMPs using infant simulators. METHODS: Expiratory airflow dispersion in term and preterm manikins was simulated (n = 288) using fine particle smoke at tidal volumes of 5 ml/kg. Using ImageJ, we quantified dispersion during common airway procedures including endotracheal tube (ETT) and T-piece ventilation. RESULTS: Maximal expiratory dispersion distances for the unsupported airway and disconnected uncuffed ETT scenarios were 30.2 and 22.7 cm (term); 22.1 and 17.2 cm (preterm), respectively. Applying T-piece positive end expiratory pressure (PEEP) via an ETT (ETTPEEP ) generated no expiratory dispersion but increased tube leak during term simulation, while ventilation breaths (ETTVENT ) caused significant expiratory dispersion and leak. There was no measurable dispersion during face mask ventilation. For term uncuffed ETT ventilation, the particle filter eliminated expiratory dispersion but increased leak. No expiratory dispersion and negligible leak were observed when combining a cuffed ETT and filter. Angulated T-pieces generated the greatest median dispersion distances of 35.8 cm (ETTPEEP ) and 23.3 cm (ETTVENT ). CONCLUSIONS: Airflow dispersion during neonatal AGMPs is greater than previously postulated and potentially could contaminate healthcare providers during resuscitation of infants infected with contagious viruses such as SARS-CoV-2. It is possible to mitigate this risk using particle filters and cuffed ETTs. Applicability in the clinical setting requires further evaluation.

Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants.

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture.

BACKGROUND: Primary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. OBJECTIVE: We compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). STUDY DESIGN: Nasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. RESULTS: Of 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. CONCLUSIONS: The overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

Streptococcus pneumoniae serotype 22F infection in respiratory syncytial virus infected neonatal lambs enhances morbidity.

Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, especially the co-pathologies between RSV and Spn, is known. Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups were mock nebulized. At day three post-RSV infection, one RSV group (RSV/Spn) and one mock-nebulized group (Spn only) were inoculated with Spn intratracheally. At day six post-RSV infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/Spn trended with higher RSV titers, but lower Spn. Additionally, lung lesions were observed to be more frequent in the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration and higher myeloperoxidase. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts.

Respiratory Virus Surveillance in Infants across Different Clinical Settings.

OBJECTIVES: We aimed to evaluate the distribution, clinical presentations and severity of common acute respiratory infections (ARI) viruses in infants across 3 clinical settings. STUDY DESIGN: In a prospective virus surveillance study, infants under 1 year with fever and/or respiratory symptoms were enrolled from outpatient, emergency department, and inpatient settings from December 16, 2019 through April 30, 2020. Demographic and clinical characteristics were collected through parent/guardian interviews, medical chart abstractions, and follow-up surveys. Nasal swabs were collected and tested for viruses using quantitative reverse-transcription polymerase chain reaction. RESULTS: We enrolled 366 infants and tested nasal swabs on 360 (98%); median age was 6.3 months, 50% male. In total, 295 (82%) had at least 1 virus detected; rhinovirus/enterovirus (RV/EV; 42%), respiratory syncytial virus (RSV; 34%), and influenza (15%) were the most common. RSV was the most frequently detected virus in the inpatient (63%) and emergency department (37%) settings, and RV/EV was most frequently detected virus in the outpatient setting (54%). RSV-positive infants had a lower median age (4.9 months) and were more likely to have respiratory distress, and RV/EV-positive infants were less likely to have respiratory distress. Influenza-positive infants had a higher median age (8 months) and were more likely to have systemic symptoms. RSV infection and younger age were associated with higher odds of hospitalization in multivariable logistic regression. CONCLUSIONS: Across 3 clinical settings, and combining virologic, patient, and health-system information, our results highlight the burden of viral ARI among infants. Overall, RSV, RV/EV, and influenza were most commonly detected, with RSV having the highest disease severity.

Seasonality of distinct respiratory viruses in a tropical city: implications for prophylaxis.

OBJECTIVE: The frequency and seasonality of viruses in tropical regions are scarcely reported. We estimated the frequency of seven respiratory viruses and assessed seasonality of respiratory syncytial virus (RSV) and influenza viruses in a tropical city. METHODS: Children (age ≤ 18 years) with acute respiratory infection were investigated in Salvador, Brazil, between July 2014 and June 2017. Respiratory viruses were searched by direct immunofluorescence and real-time polymerase chain reaction for detection of RSV, influenza A virus, influenza B virus, adenovirus (ADV) and parainfluenza viruses (PIV) 1, 2 and 3. Seasonal distribution was evaluated by Prais-Winsten regression. Due to similar distribution, influenza A and influenza B viruses were grouped to analyse seasonality. RESULTS: The study group comprised 387 cases whose median (IQR) age was 26.4 (10.5-50.1) months. Respiratory viruses were detected in 106 (27.4%) cases. RSV (n = 76; 19.6%), influenza A virus (n = 11; 2.8%), influenza B virus (n = 7; 1.8%), ADV (n = 5; 1.3%), PIV 1 (n = 5; 1.3%), PIV 3 (n = 3; 0.8%) and PIV 2 (n = 1; 0.3%) were identified. Monthly count of RSV cases demonstrated seasonal distribution (b3 = 0.626; P = 0.003). More than half (42/76 [55.3%]) of all RSV cases were detected from April to June. Monthly count of influenza cases also showed seasonal distribution (b3 = -0.264; P = 0.032). Influenza cases peaked from November to January with 44.4% (8/18) of all influenza cases. CONCLUSIONS: RSV was the most frequently detected virus. RSV and influenza viruses showed seasonal distribution. These data may be useful to plan the best time to carry out prophylaxis and to increase the number of hospital beds.