Prevalence of SARS-COV-2 and other respiratory pathogens among a Jordanian subpopulation during Delta-to-Omicron transition: Winter 2021/2022.

Acute respiratory tract infections (ARTIs) during the winter months are associated with higher morbidity and mortality compared to other seasons of the year, with children below five, elderly, and immunocompromised patients being the most susceptible. Influenza A and B viruses, rhinovirus, coronaviruses, respiratory syncytial virus, adenovirus, and parainfluenza viruses, are the most frequently identified causes of viral ARTIs. In addition, the emergence of SARS-CoV-2 in 2019 provided an additional viral cause of ARTIs. The aim of this study was to provide an overview of the epidemiological status of upper respiratory infections, their main causative agents, and reported clinical presentation in the winter months of 2021, during two important surges of COVID-19 in Jordan. Nasopharyngeal samples were collected from 339 symptomatic patients during the period from December 2021 to March 2022, followed by nucleic acid isolation using a Viral RNA/DNA extraction Kit. The causative virus species associated with the patient's respiratory symptoms was determined utilizing a multiplex real-time PCR targeting 21 viruses, 11 bacteria, and a single fungus. SARS-CoV-2 was identified in 39.2% of the patients (n = 133/339). A total of 15 different pathogens were also identified as co-infections among these 133 patients (n = 67/133). SARS-CoV-2-Bacterial coinfections (37.6%, n = 50/133) were the most frequent, with Bordetella species being the most common, followed by Staphylococcus aureus, and H.influenzae type B. Viral coinfection rate was 27.8% (n = 37/133), with Influenza B virus and Human bocavirus being the most common. In Conclusion, Both SARS-CoV-2, influenza B virus, and Bordetella accounted for the majority of infections in patients with URTI during the winter months of 2021-2022. Interestingly, more than 50% of the patients with symptoms of URTIs were confirmed to have a coinfection with two or more respiratory pathogens, with SARS-CoV-2 and Bordetella coinfection being most predominant.

Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study.

BACKGROUND: Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence. METHODS: A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1-4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets. FINDINGS: We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0-9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000-56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32-0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses. INTERPRETATION: Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications. FUNDING: CDC Foundation.

Rapid differential diagnosis of SARS-CoV-2, influenza A/B and respiratory syncytial viruses: Validation of a novel RT-PCR assay.

BACKGROUND: Influenza and respiratory syncytial (RSV) viruses are expected to co-circulate with SARS-CoV-2 in the upcoming seasons and clinical differential diagnosis between them is difficult. Laboratory-based RT-PCR is a gold standard diagnostic method for influenza, RSV and SARS-CoV-2. The objective of this study was to estimate the diagnostic performance of a novel point-of-care RT-PCR assay STANDARD M10 Flu/RSV/SARS-CoV-2 (SD Biosensor) in a large number of clinical specimens with diversified (co)-infection patterns and viral loads. METHODS: This was a retrospective study, in which all samples were tested in both STANDARD M10 Flu/RSV/SARS-CoV-2 index and Allplex SARS-CoV-2/Respiratory Panel 1 (Seegene) reference kits. Samples with discordant results were further processed in a third resolver test (Resp-4-Plex, Abbott). RESULTS: A total of 1,019 naso-/oropharyngeal samples (50.3% positive for at least one virus) were processed in both STANDARD M10 Flu/RSV/SARS-CoV-2 and Allplex assays and the overall between-assay agreement was as high as 94.6%. Positive percent agreement of the STANDARD M10 Flu/RSV/SARS-CoV-2 was 100%, 96.6%, 97.3% and 99.4% for influenza A, B, RSV and SARS-CoV-2, respectively. The corresponding negative percent agreement was 99.7%. 100%, 100% and 98.4%, respectively. The expected positive and negative predictive values for all viruses were constantly above 96% in a reasonable range of disease prevalence. CONCLUSIONS: STANDARD M10 Flu/RSV/SARS-CoV-2 is a reliable RT-PCR assay able to detect influenza A, influenza B, RSV and SARS-CoV-2 in one hour or less, fostering a rapid differential diagnosis of common respiratory viruses.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test.

The GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test (PLUS assay) received Health Canada approval in January 2022. The PLUS assay is similar to the SARS-CoV-2/Flu/RSV combination test, with modifications to improve assay robustness against circulating and emerging variants. The performance characteristics of the PLUS assay were assessed at the Lakeridge Health Oshawa Hospital Centre and the National Microbiology Laboratory of Canada. The PLUS assay was directly compared to the SARS-CoV-2/Flu/RSV combination test using SARS-CoV-2 culture from five variants and remnant clinical specimens collected across the coronavirus disease 2019 pandemic. This included 50 clinical specimens negative for all pathogens, 110 clinical specimens positive for SARS-CoV-2, influenza A, influenza B, RSVA, and(or) RSVB and an additional 11 mixed samples to screen for target interactions. The PLUS assay showed a high % agreement with the widely used SARS-CoV-2/Flu/RSV combination test. Based on these findings, the PLUS assay and the Xpert SARS-CoV-2/Flu/RSV combination test results are largely consistent with no observed difference in sensitivity, specificity, or time to result when challenged with various SARS-CoV-2 variants. The reported cycle threshold (Ct) values provided by the new PLUS assay were also unchanged, with the exception of a possible 1-2 decrease reported in Ct for RSVA across a limited sample size.

Evaluation of the cobas Liat detection test for SARS-CoV-2 and influenza viruses following the emergence of the SARS-CoV-2 Omicron variant.

OBJECTIVES: This study assessed the clinical performance of the cobas Liat SARS­CoV­2 & Influenza A/B assay (LiatCOVID/flu) for the detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses during the SARS-CoV-2 Omicron outbreak. METHODS: Residual nasopharyngeal swab samples (NPS) previously tested with cobas SARS-CoV-2 & Influenza A/B for SARS-CoV-2 and with the Allplex Respiratory Panel 1 for influenza viruses were collected. All samples were submitted to the LiatCOVID/flu assay. RESULTS: A total of 1147 samples were collected comprising 167 SARS-CoV-2-positive, 556 SARS-CoV-2-negative, 224 influenza-positive, and 200 influenza-negative cases. The positive percent agreement (PPA)/negative percent agreement (NPA) of LiatCOVID/flu for SARS-CoV-2 and influenza viruses compared to the previously tested methods were 100% of 100% and 99.6% of 100%, respectively. CONCLUSIONS: The LiatCOVID/flu assay shows an acceptable performance in the detection of SARS-CoV-2 and influenza viruses using NPS samples.

Evaluation of the Kaira COVID-19/Flu/RSV Detection Kit for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus: A comparative study with the PowerChek SARS-CoV-2, influenza A&B, RSV Multiplex Real-time PCR Kit.

BACKGROUND: Co-circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses, such as influenza and respiratory syncytial virus (RSV), can be a severe threat to public health. The accurate detection and differentiation of these viruses are essential for clinical laboratories. Herein, we comparatively evaluated the performance of the Kaira COVID-19/Flu/RSV Detection Kit (Kaira; Optolane, Seongnam, Korea) for detection of SARS-CoV-2, influenza A and B, and RSV in nasopharyngeal swab (NPS) specimens with that of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit (PowerChek; Kogene Biotech, Seoul, Korea). METHODS: A total of 250 archived NPS specimens collected for routine clinical testing were tested in parallel by the Kaira and PowerChek assays. RNA standards were serially diluted and tested by the Kaira assay to calculate the limit of detection (LOD). RESULTS: The positive and negative percent agreements between the Kaira and PowerChek assays were as follows: 100% (49/49) and 100% (201/201) for SARS-CoV-2; 100% (50/50) and 99.0% (198/200) for influenza A; 100% (50/50) and 100% (200/200) for influenza B; and 100% (51/51) and 100% (199/199) for RSV, respectively. The LODs of the Kaira assay for SARS-CoV-2, influenza A and B, and RSV were 106.1, 717.1, 287.3, and 442.9 copies/mL, respectively. CONCLUSIONS: The Kaira assay showed comparable performance to the PowerChek assay for detection of SARS-CoV-2, influenza A and B, and RSV in NPS specimens, indicating that the Kaira assay could be a useful diagnostic tool when these viruses are co-circulating.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Clinical impact of the rapid molecular detection of RSV and influenza A and B viruses in the emergency department.

INTRODUCTION: Using respiratory virus rapid diagnostic tests in the emergency department could allow better and faster clinical management. Point-of-care PCR instruments now provide results in less than 30 minutes. The objective of this study was to assess the impact of the use of a rapid molecular diagnostic test, the cobas® Influenza A/B & RSV Assay, during the clinical management of emergency department patients. METHODS: Patients (adults and children) requiring admission or suffering from an underlying condition at risk of respiratory complications were prospectively recruited in the emergency department of four hospitals in the Brussels region. Physicians' intentions regarding admission, isolation, antibiotic, and antiviral use were collected before and after performing the rapid molecular test. Additionally, a comparison of the analytical performance of this test against antigen rapid tests and viral culture was performed as well as a time-to-result evaluation. RESULTS: Among the 293 patients recruited, 90 had a positive PCR, whereas 44 had a positive antigen test. PCR yielded a sensitivity of 100% for all targets. Antigen tests yielded sensitivities ranging from 66.7% for influenza B to 83.3% for respiratory syncytial virus (RSV). The use of PCR allowed a decrease in the overall need for isolation and treatment by limiting the isolation of negative patients and antibiotic use for positive patients. Meanwhile, antiviral treatments better targeted patients with a positive influenza PCR. CONCLUSION: The use of a rapid influenza and RSV molecular test improves the clinical management of patients admitted to the emergency department by providing a fast and reliable result. Their additional cost compared to antigen tests should be balanced with the benefit of their analytical performance, leading to efficient reductions in the need for isolation and antibiotic use.

Resurgence of Influenza Circulation in the Russian Federation during the Delta and Omicron COVID-19 Era.

Influenza circulation was substantially reduced after March 2020 in the European region and globally due to the wide introduction of non-pharmaceutical interventions (NPIs) against COVID-19. The virus, however, has been actively circulating in natural reservoirs. In summer 2021, NPIs were loosened in Russia, and influenza activity resumed shortly thereafter. Here, we summarize the epidemiological and virological data on the influenza epidemic in Russia in 2021-2022 obtained by the two National Influenza Centers. We demonstrate that the commonly used baseline for acute respiratory infection (ARI) is no longer sufficiently sensitive and BL for ILI incidence was more specific for early recognition of the epidemic. We also present the results of PCR detection of influenza, SARS-CoV-2 and other respiratory viruses as well as antigenic and genetic analysis of influenza viruses. Influenza A(H3N2) prevailed this season with influenza B being detected at low levels at the end of the epidemic. The majority of A(H3N2) viruses were antigenically and genetically homogenous and belonged to the clade 3C.2a1b.2a.2 of the vaccine strain A/Darwin/9/2021 for the season 2022-2023. All influenza B viruses belonged to the Victoria lineage and were similar to the influenza B/Austria/1359417/2021 virus. No influenza A(H1N1)pdm09 and influenza B/Yamagata lineage was isolated last season.

Cepheid Xpert Xpress Flu/RSV evaluation performed by minimally trained non-laboratory operators in a CLIA-waived environment.

The COVID-19 pandemic highlighted the significance of readily available and easily performed viral testing for surveillance during future infectious pandemics. The objectives of this study were: to assess the performance of the Xpert Xpress Flu and/or RSV test, a multiplex PCR assay for detecting influenza A and B virus and respiratory syncytial virus nucleic acids in respiratory tract specimens, relative to the Quidel Lyra Influenza A+B assay and the Prodesse ProFlu+ assay, and the system's ease of use by minimally trained operators. Overall, the Xpert Xpress Flu/RSV test demonstrated a high positive and negative percent agreement with the comparator assays, and was easy to use and interpret results, based on the operators' feedback. We concluded that the Xpert Xpress Flu/RSV test is sensitive, specific, and easy to use for the diagnosis of influenza and RSV by minimally trained operators and can be a valuable tool in future infectious clusters or pandemics.

Surveillance of influenza B severe hospitalized cases during 10 seasons in Catalonia: Does the lineage make a difference?

Influenza B viruses circulate in two lineages (B/Victoria and B/Yamagata). Although classically affecting children, recently it has shown a high rate of infection and increased hospitalization in the elderly. To describe and analyze the clinical and epidemiological characteristics of severe hospitalized laboratory-confirmed influenza B virus (SHLCI-B) cases in Catalonia associated with mismatch from Influenza B virus strain included in the trivalent influenza vaccine (TIV). SHLCI-B was registered by the influenza sentinel surveillance system of Catalonia (PIDIRAC) during ten surveillance seasons from 2010 to 2020. Variables age, comorbidities, and vaccination status were recorded. Vaccine effectiveness was estimated as (1-OR) for intensive care unit (ICU) admission. Statistical significance was established at p < 0.05. A total of 1159 SHLCI-B were registered, of these 68.2% (791) corresponded to the 2017-2018 season; 21.8% (253) were admitted to ICU and 13.8% (160) were exitus; 62.5% (725) cases occurred in those aged >64 years; most frequent risk factor was cardiovascular disease (35.1%, 407) followed by chronic pulmonary obstructive disease-COPD (24.6%, 285) and diabetes (24.1%, 279). In four seasons, the predominant circulating lineage was B/Victoria, in two seasons the B/Yamagata lineage and four seasons had no IBV activity. Four seasons presented discordance with the strain included within the TIV. Vaccine effectiveness (VE) to prevent ICU admission was 31% (95% confidence interval [CI]: 4%-51%; p = 0.03); being 29% (95% CI: -3% to 51%) in discordant and 43% (95% CI:-43% to 77%) in concordant seasons. Significant differences were observed in the number of affected aged > 64 years (odds ratio [OR] = 2.5; 95% CI: 1.9-3.4; p < 0.001) and in patients with heart disease (OR = 2.40 95% CI: 1.7-3.4; p < 0.001), COPD (OR = 1.6 95% CI: 1.1-2.3; p = 0.01), and diabetes (OR = 1.5 95% CI: 1.1-2.1; p = 0.04) between discordant and concordant seasons. The increase in hospitalization rate in people> 64 years of age and those presenting comorbidities in seasons with circulating influenza B virus belonging to a lineage discordant with the strain included in the TIV and the decrease of VE to prevent ICU admissions evidence the vital need to administer the quadrivalent influenza vaccine regardless of the findings of predominant circulation in the previous season.

Influenza Activity and Composition of the 2022-23 Influenza Vaccine - United States, 2021-22 Season.

Before the emergence of SARS-CoV-2, the virus that causes COVID-19, influenza activity in the United States typically began to increase in the fall and peaked in February. During the 2021-22 season, influenza activity began to increase in November and remained elevated until mid-June, featuring two distinct waves, with A(H3N2) viruses predominating for the entire season. This report summarizes influenza activity during October 3, 2021-June 11, 2022, in the United States and describes the composition of the Northern Hemisphere 2022-23 influenza vaccine. Although influenza activity is decreasing and circulation during summer is typically low, remaining vigilant for influenza infections, performing testing for seasonal influenza viruses, and monitoring for novel influenza A virus infections are important. An outbreak of highly pathogenic avian influenza A(H5N1) is ongoing; health care providers and persons with exposure to sick or infected birds should remain vigilant for onset of symptoms consistent with influenza. Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.

Using amino acid features to identify the pathogenicity of influenza B virus.

BACKGROUND: Influenza B virus can cause epidemics with high pathogenicity, so it poses a serious threat to public health. A feature representation algorithm is proposed in this paper to identify the pathogenicity phenotype of influenza B virus. METHODS: The dataset included all 11 influenza virus proteins encoded in eight genome segments of 1724 strains. Two types of features were hierarchically used to build the prediction model. Amino acid features were directly delivered from 67 feature descriptors and input into the random forest classifier to output informative features about the class label and probabilistic prediction. The sequential forward search strategy was used to optimize the informative features. The final features for each strain had low dimensions and included knowledge from different perspectives, which were used to build the machine learning model for pathogenicity identification. RESULTS: The 40 signature positions were achieved by entropy screening. Mutations at position 135 of the hemagglutinin protein had the highest entropy value (1.06). After the informative features were directly generated from the 67 random forest models, the dimensions for class and probabilistic features were optimized as 4 and 3, respectively. The optimal class features had a maximum accuracy of 94.2% and a maximum Matthews correlation coefficient of 88.4%, while the optimal probabilistic features had a maximum accuracy of 94.1% and a maximum Matthews correlation coefficient of 88.2%. The optimized features outperformed the original informative features and amino acid features from individual descriptors. The sequential forward search strategy had better performance than the classical ensemble method. CONCLUSIONS: The optimized informative features had the best performance and were used to build a predictive model so as to identify the phenotype of influenza B virus with high pathogenicity and provide early risk warning for disease control.

Influenza returns with a season dominated by clade 3C.2a1b.2a.2 A(H3N2) viruses, WHO European Region, 2021/22.

In the WHO European Region, COVID-19 non-pharmaceutical interventions continued slowing influenza circulation in the 2021/22 season, with reduced characterisation data. A(H3) predominated and, in some countries, co-circulated with A(H1)pdm09 and B/Victoria viruses. No B/Yamagata virus detections were confirmed. Substantial proportions of characterised circulating virus subtypes or lineages differed antigenically from their respective northern hemisphere vaccine components. Appropriate levels of influenza virus characterisations should be maintained until the season end and in future seasons, when surveillance is adapted to integrate SARS-CoV-2.

Tracheal Aspirate and Bronchoalveolar Lavage as Potential Specimen Types for COVID-19 Testing Using the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV.

Xpert Xpress SARS-CoV-2/Flu/RSV is a rapid diagnostic test currently approved for the detection of SARS-CoV-2 using upper respiratory tract specimens. This study attempts to assess the performance of this assay using upper and lower respiratory tract specimens by comparing its results to the lab-developed PCR test. We assessed the performance of GeneXpert for the detection of SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus for upper respiratory tract specimens. In addition, the SARS-CoV-2 detection was evaluated for lower respiratory tract specimens (bronchoalveolar lavage and tracheal aspirate). Precision and reproducibility of the test were also assessed using samples with varying cycle threshold values. Xpert Xpress SARS-CoV-2/Flu/RSV shows 100% positive and negative agreements for all four targets when tested using upper respiratory tract specimens. For lower respiratory tract specimens, tracheal aspirate and bronchoalveolar lavage samples respectively show 96% and 100% positive percent agreement for SARS-Cov-2 target only. No positive flu/RSV samples were included for lower respiratory tract specimens. Both samples show 100% negative percent agreement. The precision and reproducibility assay also showed 100% correspondence. Xpert Xpress SARS-CoV-2/Flu/RSV can be potentially used for SARS-Cov-2 detection in lower respiratory tract specimens. Performance passed our study acceptance criteria and shows promising implications as a point of care detection assay. IMPORTANCE Cepheid's Xpert Xpress SARS-CoV-2/Flu/RSV provides a means of rapid diagnosis that can help in hospital bed management and patient flow. It is also important for each microbiology lab to increase its capacity and most importantly have a different platform to overcome the anticipated reagent shortage at times of peak community transmission. There is limited evidence on using it for lower respiratory tract specimens. Here we present our validation for upper respiratory tract specimens as well as a potential use for lower respiratory specimens (BAL and TA), and we discuss some of the applications we have been using in our organization.

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput.

During the COVID-19 pandemic, sample pooling has proven an effective strategy to overcome the limitations of reagent shortages and expand laboratory testing capacity. The inclusion of influenza and respiratory syncytial virus (RSV) in a multiplex tandem PCR platform with SARS-CoV-2 provides useful diagnostic and infection control information. This study aimed to evaluate the performance of the influenza and RSV targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay, including the effect of pooling samples on target detection. RSV target detection in clinical samples was compared to the Cepheid Xpert Xpress Flu/RSV assay as a reference standard. Samples were then tested in pools of four and detection rates were compared. Owing to the unavailability of clinical samples for influenza, only the effect of sample pooling on simulated samples was evaluated for these targets. RSV was detected in neat clinical samples with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.5% compared to the reference standard, demonstrating 99.7% agreement. This study demonstrates that sample pooling by four increases the average Ct value by 2.24, 2.29, 2.20 and 1.91 cycles for the target's influenza A, influenza A typing, influenza B and RSV, respectively. The commercial AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay was able to detect influenza and RSV at an intermediate concentration within the limit of detection of the assay. Further studies to explore the applicability of sample pooling at the lower limit of detection of the assay is needed. Nevertheless, sample pooling has shown to be a viable strategy to increase testing throughput and reduce reagent usage. In addition, the multiplexed platform targeting various respiratory viruses assists with public health and infection control responses, clinical care, and patient management.

Evaluation of Three Multiplex Real-time Reverse Transcription PCR Assays for Simultaneous Detection of SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus in Nasopharyngeal Swabs.

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.

No evidence of SARS-CoV-2 in hospitalized patients with severe acute respiratory syndrome in five Italian hospitals from 1st November 2019 to 29th February 2020.

BACKGROUND: On 9th January 2020, China CDC reported a novel coronavirus (later named SARS-CoV-2) as the causative agent of the coronavirus disease 2019 (COVID-19). Identifying the first appearance of virus is of epidemiological importance to tracking and mapping the spread of SARS-CoV-2 in a country. We therefore conducted a retrospective observational study to detect SARS-CoV-2 in oropharyngeal samples collected from hospitalized patients with a Severe Acute Respiratory Infection (SARI) enrolled in the DRIVE (Development of Robust and Innovative Vaccine Effectiveness) study in five Italian hospitals (CIRI-IT BIVE hospitals network) (1st November 2019 - 29th February 2020). OBJECTIVES: To acquire new information on the real trend in SARS-CoV-2 infection during pandemic phase I and to determine the possible early appearance of the virus in Italy. MATERIALS AND METHODS: Samples were tested for influenza [RT-PCR assay (A/H1N1, A/H3N2, B/Yam, B/Vic)] in accordance with the DRIVE study protocol. Subsequently, swabs underwent molecular testing for SARS-COV-2. [one-step real-time multiplex retro-transcription (RT) PCR]. RESULTS: In the 1683 samples collected, no evidence of SARS-CoV-2 was found. Moreover, 28.3% (477/1683) of swabs were positive for influenza viruses, the majority being type A (358 vs 119 type B). A/H3N2 was predominant among influenza A viruses (55%); among influenza B viruses, B/Victoria was prevalent. The highest influenza incidence rate was reported in patients aged 0-17 years (40.3%) followed by those aged 18-64 years (24.4%) and ≥65 years (14.8%). CONCLUSIONS: In Italy, some studies have shown the early circulation of SARS-CoV-2 in northern regions, those most severely affected during phase I of the pandemic. In central and southern regions, by contrast no early circulation of the virus was registered. These results are in line with ours. These findings highlight the need to continue to carry out retrospective studies, in order to understand the epidemiology of the novel coronavirus, to better identify the clinical characteristics of COVID-19 in comparison with other acute respiratory illnesses (ARI), and to evaluate the real burden of COVID-19 on the healthcare system.

Verification of the Abbott Alinity m Resp-4-Plex assay for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus.

COVID-19 symptomology may overlap with other circulating respiratory viruses that may also cause severe disease and for which there are specific and potentially life-saving treatments. The Abbott Alinity m Resp-4-Plex assay is a multiplex PCR assay that simultaneously detects and differentiates infection with SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). We characterized its accuracy, precision, and analytical sensitivity. All were found to be robust for measures examined. In the context of sample-to-answer, near random access automation on the Alinity m platform, we believe that the Resp-4-Plex assay offers significant utility in addressing the current needs of the SARS-CoV-2 pandemic and future needs during anticipated endemic circulation of SARS-CoV-2 with other respiratory viruses.

Comparison of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit and BioFire Respiratory Panel 2.1 for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus.

The potential co-circulation of SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) could pose an unprecedented challenge to healthcare systems worldwide. Here, we compared the performance of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit (PowerChek) for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus with that of BioFire Respiratory Panel 2.1 (RP2.1) using 175 nasopharyngeal swab (NPS) specimens. Positive percent agreement and negative percent agreement of the PowerChek assay compared to RP2.1 were as follows: 100 % (40/40) and 100 % (135/135) for SARS-CoV-2; 100 % (39/39) and 100 % (136/136) for influenza A; 100 % (35/35) and 100 % (140/140) for influenza B; and 93.1 % (27/29) and 100 % (146/146) for RSV, respectively. The limit of detection (LOD) was accessed using RNA standards for each virus, and the LOD values of the PowerChek assay for SARS-CoV-2, influenza A and B, and RSV were 0.36, 1.24, 0.09, and 0.63 copies/µL, respectively. Our results demonstrate that the PowerChek assay is sensitive and accurate for detection of SARS-CoV-2, influenza A and B, and RSV, suggesting that this assay can be a valuable diagnostic tool when SARS-CoV-2, influenza, and RSV are co-circulating.