Detection Of Respiratory Viruses Research Articles

3D Film-Like Nanozyme with a Synergistic Amplification Effect for the Ultrasensitive Immunochromatographic Detection of Respiratory Viruses.

Greatly improving the sensitivity and detection range of lateral flow immunoassays (LFAs) by at least 100 times without using additional instruments remains challenging. Herein, we develop a three-dimensional (3D) film-like nanozyme (GO-Pt30-AuPt5) by ordered assembly of one layer of 30 nm Pt nanoparticles (NPs) and one layer of small Au@Pt satellites (5 nm) onto a two-dimensional (2D) graphene oxide (GO) nanofilm, in which GO greatly increased the interface area and stability of the nanozyme whereas Pt and Au@Pt NPs synergistically enhanced colorimetric/catalytic activities. The grafting of outer Au@Pt satellites converted the 2D nanofilm into a 3D flexible nanozyme with numerous catalytic sites for enzymatic deposition signal amplification and binding sites for target capture. The introduction of GO-Pt30-AuPt5 into multiplex LFA achieved the ultrasensitive and simultaneous detection of two important respiratory viruses with sensitivity of 1 pg/mL level, which was about 100 times higher than that without signal enrichment and at least 20 and 1900 times higher than those of traditional enzyme-linked immunosorbent assay and AuNP-based LFA, respectively. The clinical utility of the proposed assay was validated through the diagnosis of 49 real clinical respiratory tract specimens. Our proposed LFA shows great potential for the ultrasensitive screening of pathogens in the field.

Prevalence and Seasonal Patterns of 16 Common Viral Respiratory Pathogens during the COVID-19 Pandemic in Gauteng Province, South Africa, 2020-2021.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic resulted in widespread morbidity and mortality, but generally, the diagnosis of other respiratory viruses was limited. This study aimed to assess the prevalence of other respiratory viruses during the 2020/2021 pandemic among patients of all ages who accessed care at public healthcare facilities in Gauteng Province, South Africa. Laboratory diagnosis for respiratory viruses, with or without SARS-CoV-2, was conducted via multiplex real-time polymerase chain reactions using respiratory specimens. A total of 1776 patients were included from 1 April 2020 to 31 March 2021, of which 766 (43.1%) were positive for respiratory viruses other than SARS-CoV-2. RV (368/1776; 20.7%) was the most prevalent, followed by RSV (304/1776; 17.1%), AdV (112/1776; 6.3%) and EV (105/1776; 5.9%). hCoV-OC43 (39/1776; 2.2%) was the most prevalent common coronavirus. SARS-CoV-2 co-infections were detected in 4.8% (24/500) of patients. Only 27.1% (482/1776) of patients were admitted to high-care or intensive care units. A decrease in respiratory virus detections was observed, except for RSV, EV and hCoV-OC43. RSV prevalence increased in 2021, while influenza A/B viruses remained undetected.

Molecular Detection of Respiratory Tract Viruses in Chickens at the Point of Need by Loop-Mediated Isothermal Amplification (LAMP).

Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP's main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need.

Respiratory viral infections in children with cancer and febrile neutropenia and children undergoing hematopoietic stem cell transplantation.

The scope of this review is to understand the epidemiology and potential role of respiratory viral infections in children with cancer and febrile neutropenia, as well as in children, undergoing hematopoietic stem cell transplantation. Early detection of respiratory viral infections through molecular diagnostic techniques has allowed recent randomized clinical studies to advance the possibility of more rational use of antimicrobials in this susceptible population. Progress has been made in the early detection of respiratory viruses in episodes of fever and neutropenia in children with cancer. In selected patients who meet specific clinical safety criteria and have negative bacterial cultures, it has been possible to safely and effectively discontinue antimicrobials. This has been validated in recent randomized clinical studies. However, more evidence is still needed for a similar indication in children, undergoing hematopoietic stem cell transplantation with viral respiratory infection episodes. Understanding the role of respiratory viral infections in populations of immunocompromised children may contribute to a more rational use of antimicrobials and, in the near future, may help to decrease antimicrobial resistance in this susceptible population.

Epidemiological study of post-pandemic pediatric common respiratory pathogens using multiplex detection

BackgroundThe burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system.MethodsSix types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples.ResultsAmong the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P < 0.001), and were more common in younger children. PIV-1 was more common in infants, toddlers, and preschoolers than in school-age children (P < 0.001). FluB was predominantly detected in school-age children (P < 0.001). RSV-, ADV-, and PIV-1-positivity rates were higher in 2022 than in 2023. Seasonal viral patterns differed across years. RSV (OR 9.156. 95% CI 5.905–14.195) and PIV-3 (OR 1.683, 95% CI 1.133–2.501) were risk factors for lower respiratory tract infections. RSV-positivity was associated with severe pneumonia (P = 0.044). PIV-3 (OR 0.391, 95% CI 0.170–0.899), summer season (OR 1.982, 95% CI 1.117–3.519), and younger age (OR 0.938, 95% CI 0.893–0.986) influenced pneumonia severity. Multiplex RT-qPCR showed good diagnostic performance.ConclusionAfter changes in COVID-19 prevention and control strategies, six common respiratory viruses in children were prevalent in 2022–2023, with different seasonal epidemic characteristics and age proclivities. RSV and PIV-3 cause lower, and FluA, FluB, and ADV more typically cause upper respiratory tract infections. Infancy and summer season influence severe pneumonia risk. Multiplex RT-qPCR is valuable for accurate and timely detection of respiratory viruses in children, which facilitates management, treatment, and prevention of ARTIs.

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

School knowledge of infectious diseases in schools: conducting surveillance and on-demand, symptomatic respiratory viral testing in a large pre-kindergarten-12th grade school district.

Limited data about acute respiratory illness (ARI) and respiratory virus circulation are available in congregate community settings, specifically schools. To better characterize the epidemiology of ARI and respiratory viruses in schools, we developed School Knowledge of Infectious Diseases in Schools (School KIDS). School KIDS is a prospective, respiratory viral testing program in a large metropolitan school district (pre-kindergarten-12th grade) in Kansas City, Missouri. During the 2022-2023 school year, all students and staff were eligible to participate in surveillance respiratory viral testing at school by submitting observed self-administered nasal swabs monthly. Participants could also submit a nasal swab for on-demand symptomatic testing when experiencing ≥1 ARI symptom, including cough, fever, nasal congestion, runny nose, shortness of breath, sore throat, and/or wheezing. Swabs were tested in a research laboratory using multipathogen respiratory polymerase chain reaction assays. Participants were evaluated for ongoing viral shedding by collecting two weekly nasal swabs (i.e., convalescent), following initial on-demand symptomatic testing. Participants were asked to complete an electronic survey to capture the presence and type of ARI symptom(s) before the collection of respiratory swabs. From 31 October 2022 to 29 June 2023, School KIDS enrolled 978 participants, including 700 students, representing 3.4% of the district student population, and 278 staff members. Participants submitted a median of six surveillance, one symptomatic, and two convalescent specimens during the study period. A total of 6,315 respiratory specimens, including 4,700 surveillance, 721 on-demand symptomatic, and 894 convalescent specimens, were tested. Overall, a virus was detected in 1,168 (24.9%) surveillance and 363 (50.3%) symptomatic specimens. Of the 5,538 symptom surveys sent to participants before scheduled surveillance testing, 4,069 (73.5%) were completed; ARI symptoms were reported on 1,348 (33.1%) surveys. Respiratory surveillance testing in schools is feasible and provides novel information about respiratory virus detections in students and staff attending school. Schools are an important community setting, and better knowledge of respiratory virus circulation in schools may be useful to identify respiratory virus transmission in the community and assess the impact of effective infection prevention measures.

Changes in the etiological structure of severe acute respiratory viral infections in children and adults under the influence of the COVID-19 pandemic

Introduction. The traditional surveillance system for influenza and ARVI provides a general description of epidemics, but does not provide information on the age-related characteristics of the etiology and clinical peculiarities of severe acute respiratory diseases (SARI) in hospitalized patients. Aim. To monitor the etiology of SARI in hospitalized children and adults, assessing the impact of the COVID-19 pandemic on this process. Materials and methods. Standardized clinical and laboratory monitoring of SARI among 18,458 hospitalized patients was carried out in hospitals in three cities of Russia with weekly PCR detection of 11 types of pathogens. Results. According to the investigation of hospitalized patients with SARI for the period from 2018 to 2023, the viral etiology of respiratory diseases was deciphered in 58.3% of cases. Weekly monitoring showed a change in the etiological mosaic of SARI pathogens during the SARS-CoV-2 pandemic with a sharp decrease in the frequency of detection of influenza and respiratory syncytial virus (RSV) during the 2020–2021 season against the background of a significant increase of metapneumovirus and rhinovirus infections in children. During the 2022-2023 season an increase in the proportion of RSV infection in children under 6 years of age (up to 36.2%) was noted against the background of a significant decrease in the frequency of SARS-CoV-2. In the intensive care units (ICU), RSV infection was most often in children during the post-pandemic period (up to 30.1–53.6% of positive cases, p 0.001); in adults, SARS-CoV-2 was mostly detected (76,5–100% of cases, p 0.001). Conclusion. Hospital surveillance data significantly complements the epidemiological information obtained in the traditional surveillance system. Monitoring of infections has shown a continuously changing etiological infrastructure of SARI, with the disappearance of influenza and RSV during the COVID-19 pandemic and their return to circulation in the post-pandemic period.

Development of a label-free impedimetric immunosensor for the detection of respiratory syncytial virus

Development of a label-free impedimetric immunosensor for the detection of respiratory syncytial virus

One-base-mismatch CRISPR-based transistors for single nucleotide resolution assay

An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 μL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 105 copy mL–1 in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 μL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics.

Pathogenic features and clinical characteristics of acute community-acquired lower respiratory tract infections.

To investigate the pathogen distribution and clinical characteristics of acute community-acquired lower respiratory tract infections (CALRTIs). This was a retrospective study. The clinical data of 218 patients with CALRTIs admitted to Baoding No.1 Central Hospital from December 2021 to December 2022 were retrospectively collected and were divided into two groups according to the results of polymerase chain reaction(PCR) testing using a nasopharyngeal swab: streptococcus pneumoniae positive group(observation group) and non-streptococcus pneumoniae positive group(control group). Clinical symptoms, blood gas analysis indicators were compared between the two groups. Haemophilus influenzae and Staphylococcus aureus, as well as virus and atypical pathogen infection, were the predominant pathogenic bacteria in both groups. No statistically significant differences were observed in the positive rates of sputum smear, sputum culture, respiratory virus detection and atypical pathogen detection between the two groups(P>0.05). However, the control group had a higher detection rate of gram-positive bacteria, gram-negative bacteria and Legionella pneumophila in sputum smears than the observation group, with a statistically significant difference(P<0.05). One death occurred in each group, with no significant difference in mortality and six in each group left the hospital or were transferred due to deterioration, with no significant difference in improved discharge rates. Acute community-acquired lower respiratory tract infections(CALRTIs) take bacteria, viruses and atypical pathogens as its leading pathogenic bacteria. In the treatment of patients with acute CALRTIs, early pathogenic examination should be performed to assist in guiding antibiotic therapy for rapid control, early recovery and ameliorated clinical outcomes.

Coinfections and comorbidities observed in COVID-19 during the influenza season in the pediatric patient.

To evaluate if the comorbidity and coinfections presented by SARS-CoV-2 infection vs. COVID-19 impact our Mexican children. Prospective and observational study that included the 2020-2021 peak influenza season. All patients with a diagnosis of infection by SARS-CoV-2 vs. COVID-19 who were admitted to the Hospital Infantil de Mexico were analyzed. Real-time RT-PCR for SARS-CoV-2 was performed in all patients, determining E, RdRp and RP genes and protein N, as well as RT-PCR for detection of respiratory viruses. The inclusion criteria were met by 163 patients. The group with the highest risk of becoming ill was adolescents (40.4%), followed by schoolchildren and preschoolers (21.4% and 19.6% of the cases, respectively). There were three cases with viral coinfection: two (1.2%) with parvovirus B-19 and one (0.6%) with herpes type I; another two (1.2%) showed bacterial coinfection. The main comorbidity were obesity, acute lymphoblastic leukemia and arterial hypertension. Regarding mortality, we only had four cases (2.4%). Obesity, cancer, hypertension, heart disease and diabetes are comorbidity present in our patients, as referred to in literature, but not coinfections. In our study, we did not have any associated mortality related to comorbidity.

Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era

BackgroundThe concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting.MethodsTwo time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2.ResultsThe results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022.ConclusionThe results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Multiplexed detection of eight respiratory viruses based on nanozyme colorimetric microfluidic immunoassay.

Pandemics caused by respiratory viruses, such as the SARS-CoV-1/2, influenza virus, and respiratory syncytial virus, have resulted in serious consequences to humans and a large number of deaths. The detection of such respiratory viruses in the early stages of infection can help control diseases by preventing the spread of viruses. However, the diversity of respiratory virus species and subtypes, their rapid antigenic mutations, and the limited viral release during the early stages of infection pose challenges to their detection. This work reports a multiplexed microfluidic immunoassay chip for simultaneous detection of eight respiratory viruses with noticeable infection population, namely, influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, human bocavirus, human metapneumovirus, adenovirus, and human parainfluenza viruses. The nanomaterial of the nanozyme (Au@Pt nanoparticles) was optimized to improve labeling efficiency and enhance the detection sensitivity significantly. Nanozyme-binding antibodies were used to detect viral proteins with a limit of detection of 0.1pg/mL with the naked eye and a microplate reader within 40min. Furthermore, specific antibodies were screened against the conserved proteins of each virus in the immunoassay, and the clinical sample detection showed high specificity without cross reactivity among the eight pathogens. In addition, the microfluidic chip immunoassay showed high accuracy, as compared with the RT-PCR assay for clinical sample detection, with 97.2%/94.3% positive/negative coincidence rates. This proposed approach thus provides a convenient, rapid, and sensitive method for simultaneous detection of eight respiratory viruses, which is meaningful for the early diagnosis of viral infections. Significantly, it can be widely used to detect pathogens and biomarkers by replacing only the antigen-specific antibodies.

The Changing Landscape of Respiratory Viruses Contributing to Hospitalizations in Quebec, Canada: Results From an Active Hospital-Based Surveillance Study.

A comprehensive description of the combined effect of SARS-CoV-2 and respiratory viruses other than SARS-CoV-2 (ORVs) on acute respiratory infection (ARI) hospitalizations is lacking. This study aimed to compare the viral etiology of ARI hospitalizations before the pandemic (8 prepandemic influenza seasons, 2012-13 to 2019-20) and during 3 pandemic years (periods of increased SARS-CoV-2 and ORV circulation in 2020-21, 2021-22, and 2022-23) from an active hospital-based surveillance network in Quebec, Canada. We compared the detection of ORVs and SARS-CoV-2 during 3 pandemic years to that in 8 prepandemic influenza seasons among patients hospitalized with ARI who were tested systematically by the same multiplex polymerase chain reaction (PCR) assay during periods of intense respiratory virus (RV) circulation. The proportions of infections between prepandemic and pandemic years were compared by using appropriate statistical tests. During prepandemic influenza seasons, overall RV detection was 92.7% (1384/1493) (respiratory syncytial virus [RSV]: 721/1493, 48.3%; coinfections: 456/1493, 30.5%) in children (<18 years) and 62.8% (2723/4339) (influenza: 1742/4339, 40.1%; coinfections: 264/4339, 6.1%) in adults. Overall RV detection in children was lower during pandemic years but increased from 58.6% (17/29) in 2020-21 (all ORVs; coinfections: 7/29, 24.1%) to 90.3% (308/341) in 2021-22 (ORVs: 278/341, 82%; SARS-CoV-2: 30/341, 8.8%; coinfections: 110/341, 32.3%) and 88.9% (361/406) in 2022-23 (ORVs: 339/406, 84%; SARS-CoV-2: 22/406, 5.4%; coinfections: 128/406, 31.5%). In adults, overall RV detection was also lower during pandemic years but increased from 43.7% (333/762) in 2020-21 (ORVs: 26/762, 3.4%; SARS-CoV-2: 307/762, 40.3%; coinfections: 7/762, 0.9%) to 57.8% (731/1265) in 2021-22 (ORVs: 179/1265, 14.2%; SARS-CoV-2: 552/1265, 43.6%; coinfections: 42/1265, 3.3%) and 50.1% (746/1488) in 2022-23 (ORVs: 409/1488, 27.5%; SARS-CoV-2: 337/1488, 22.6%; coinfections: 36/1488, 2.4%). No influenza or RSV was detected in 2020-21; however, their detection increased in the 2 subsequent years but did not reach prepandemic levels. Compared to the prepandemic period, the peaks of RSV hospitalization shifted in 2021-22 (16 weeks earlier) and 2022-23 (15 weeks earlier). Moreover, the peaks of influenza hospitalization shifted in 2021-22 (17 weeks later) and 2022-23 (4 weeks earlier). Age distribution was different compared to the prepandemic period, especially during the first pandemic year. Significant shifts in viral etiology, seasonality, and age distribution of ARI hospitalizations occurred during the 3 pandemic years. Changes in age distribution observed in our study may reflect modifications in the landscape of circulating RVs and their contribution to ARI hospitalizations. During the pandemic period, SARS-CoV-2 had a low contribution to pediatric ARI hospitalizations, while it was the main contributor to adult ARI hospitalizations during the first 2 seasons and dropped below ORVs during the third pandemic season. Evolving RVs epidemiology underscores the need for increased scrutiny of ARI hospitalization etiology to inform tailored public health recommendations.

Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Clinical Performance of Self-Collected Purified Water Gargle for Detection of Influenza a Virus Infection by Real-Time RT-PCR.

Self-collected specimens are increasingly being used as alternatives to swab-based methods for the detection of respiratory viruses. While saliva is well accepted, gargle specimens are a potential alternative with characteristics that are more favorable for laboratory handling. This study assessed the performance of gargle specimens in the detection of influenza A viruses (IAVs). We performed a prospective head-to-head comparison between combined nasopharyngeal and oropharyngeal swabs (NPS&OPS) and purified water gargle (PWG) among adult outpatients with febrile respiratory symptoms to detect IAVs using real-time RT-PCR during two influenza seasons. During study periods 1 (July 13 to 26, 2022, H3N2 predominated) and 2 (February 25 to March 10, 2023, H1N1 pdm09 predominated), a total of 459 patients were recruited. The overall agreement between the NPS&OPS and PWG was 85.0% (390/459, κ = 0.697), with 88.0% in period 1 and 82.6% in period 2. The detection rate of IAVs in PWG (51.6%, 237/459) was lower than that in NPS&OPS (62.3%, 286/459) (p < 0.0001). The overall sensitivity and specificity were 96.6% (93.7-98.3%) and 100% (97.1-100%) in NPS&OPS and were 80.1% (75.0-84.4%) and 100% (97.1-100%) in PWG, respectively. Among the 227 pairs of concordant positive specimens, cycle threshold (Ct) values were significantly lower in NPS&OPS than in PWG (median Ct values: 24.2, 28.2, p < 0.0001). Although self-collected PWG specimens offer acceptable performance for IAVs molecular testing, NPS&OPS remain a reliable option. Given the convenience of collection, nonviscous gargles are recommended for viral detection during emergencies or under specific conditions.

Frequency of detection of respiratory viruses (Infuenza A/B, RSV) among admitted patients in a tertiary hospital

Frequency of detection of respiratory viruses (Infuenza A/B, RSV) among admitted patients in a tertiary hospital

Frequency of respiratory virus detection in the upper respiratory tract of hospitalized patients

Frequency of respiratory virus detection in the upper respiratory tract of hospitalized patients