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

BACKGROUND: The 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. METHODS: Two 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. RESULTS: The 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. CONCLUSION: The 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.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Molecular characterization of influenza virus circulating in Nepal in the year 2019.

Influenza (sometimes referred to as "flu") is a contagious viral infection of the airways in the lungs that affects a significant portion of the world's population. Clinical symptoms of influenza virus infections can range widely, from severe pneumonia to moderate or even asymptomatic sickness. If left untreated, influenza can have more severe effects on the heart, brain, and lungs than on the respiratory tract and can necessitate hospitalization. This study was aimed to investigate and characterize all types of influenza cases prevailing in Nepal and to analyze seasonal occurrence of Influenza in Nepal in the year 2019. A cross sectional, retrospective and descriptive study was carried out at National Influenza Center (NIC), National Public Health Laboratory Kathmandu Nepal for the period of one year (Jan-Dec 2019). A total of 3606 throat swab samples from various age groups and sexes were processed at the NIC. The specimens were primarily stored at 4 °C and processed using ABI 7500 RT PCR system for the identification of Influenza virus types and subtypes. Data accessed for research purpose were retrieved from National Influenza Centre (NIC) on 1st Jan 2020. Of the total 3606 patients suspected of having influenza infection, influenza viruses were isolated from 1213 (33.6%) patients with male predominance. The highest number of infection was caused by Influenza A/Pdm09 strain 739 (60.9%) followed by Influenza B 304 (25.1%) and Influenza A/H3 169 (13.9%) and most remarkable finding of this study was the detection of H5N1 in human which is the first ever case of such infection in human from Nepal. Similar to other tropical nations, influenza viruses were detected year-round in various geographical locations of Nepal. The influenza virus type and subtypes that were in circulation in Nepal were comparable to vaccine candidate viruses, which the currently available influenza vaccine may prevent.

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.

Effect of combined infection with Salmonella and influenza virus on their respective proliferation in chicken embryonated eggs.

Background: Salmonella is a major food-borne bacterial pathogen that causes food poisoning related to the consumption of eggs, milk, and meat. Food safety in relation to Salmonella is particularly important for eggs because their shells as well as their contents can be a source of contamination. Chicken can also be infected with influenza virus, but it remains unclear how co-infection of Salmonella and influenza virus affect each other. Aim: The potential influence of co-infection of Salmonella and influenza virus was examined. Methods: Salmonella Abony and influenza virus were injected into chicken embryonated eggs. After incubation, proliferation of Salmonella and influenza virus was measured using a direct culture assay for bacteria and an enzyme-linked immunosorbent assay for influenza virus, respectively. Results: Our findings indicate that the number of colony-forming units (CFUs) of Salmonella did not vary between chicken embryonated eggs co-infected with influenza A virus and Salmonella-only infected eggs. Furthermore, we found the proliferation of influenza A or B virus was not significantly influenced by co-infection of the eggs with Salmonella. Conclusion: These results suggest that combined infection of Salmonella with influenza virus does not affect each other, at least in terms of their proliferation.

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.

Evaluation of the analytical and clinical performance of two RT-PCR based point-of-care tests; Cepheid Xpert® Xpress CoV-2/Flu/RSV plus and SD BioSensor STANDARD™ M10 Flu/RSV/SARS-CoV-2.

BACKGROUND: Rapid and accurate detection of viral respiratory infections is important for infection control measures. This study compares the analytical and clinical performance of the Xpert® Xpress CoV-2/Flu/RSV plus test ("Xpert", Cepheid) and the STANDARD™ M10 Flu/RSV/SARS-CoV-2 test ("M10", SD Biosensor). Both tests are quadruplex RT-PCR assays for rapid diagnosis of SARS-CoV-2, influenza A/B and RSV. STUDY DESIGN: Analytical sensitivities were determined by limit of detection for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Additionally, the clinical performance of the Xpert and the M10 tests was evaluated against standard-of-care RT-PCR by testing of 492 clinical specimens. RESULTS: The analytical sensitivities for Xpert versus M10 test was 10, 50, 50 and 300 versus 300, 200, 800 and 1500 copies/mL for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Clinical sensitivity for the Xpert test was superior across all four pathogens compared to the M10 test. Xpert showed clinical sensitivity of 100 % in all Ct-ranges for all four pathogens whereas M10 showed clinical sensitivity of 100 % in the 25-30 Ct-range, 84-100 % in the 30-35 Ct-range and 47-67 % in the >35 Ct-range across the four pathogens. Translating into real-life clinical sensitivity, the Xpert would detect 100 % of all four pathogens, whereas M10 would detect 92.1, 92.4, 84.8 and 94.7 % for SARS-CoV-2, influenza A, influenza B and RSV. CONCLUSION: This study demonstrates improved analytical and clinical performance of Xpert Xpress CoV-2/Flu/RSV plus compared to STANDARD M10 Flu/RSV/SARS-CoV-2, which is important for ensuring accuracy of diagnosis at all stages of a respiratory infection.

Evaluation of CLINITEST® Rapid Covid-19 + Influenza antigen test in a cohort of symptomatic patients in an emergency department.

OBJECTIVES: Rapid management of patients with respiratory tract infections in hospital emergency departments is one of the main objectives since the concurrent circulation of respiratory viruses following the SARS-CoV-2 pandemic. The use of new combined point-of-care antigen tests for detecting influenza A/B and SARS-CoV-2 represents an advantage in response time over the molecular tests. The objective was to evaluate the suitability of the CLINITEST® Rapid Covid-19 + Influenza Antigen test (Siemens Healthineers, Germany) (RCIA test) by measuring the sensitivity, specificity, Cohen's kappa, and cut-off values. METHODS: Nasopharyngeal samples were collected from a randomised group of symptomatic patients of all ages at emergency department during January-February 2023. In parallel, these patients were screened for influenza A/B, and SARS-CoV-2 using RT-PCR. The Ct (cycle threshold) values were collected for positive [RT-PCR (+) /RCIA test (+)] and false negative [(RT-PCR (+) /RCIA test (-)] samples. A subanalysis was performed in the paediatric population (< 16 years-old). RESULTS: We included 545 patients (55.8% females) with a median age of 7 years-old (IQR: 1-66.5). The RCIA test showed a sensitivity of 59.7% [95%CI: 46.9-67.33] for influenza A, 65.6% [95%CI: 49.5-80.3] for influenza B, and 76.9% [95%CI: 45.8-84.8] for SARS-CoV-2. The specificity was between 90.7%-99.7% with a moderate/high level of agreement with RT-PCR (kappa score: 0.6-0.8) for the three respiratory viruses included in the RCIA test. CONCLUSIONS: The sensitivity of the RCIA test is insufficient for screening of patients, including patients with low Ct values (Ct > 20). Despite its good specificity and Cohen's kappa value, its use as a screening test is not comparable to RT-PCR systems in the ED environment with a high number of false negative results.

Multiplexed electrochemical liposomes applied to the detection of nucleic acids for Influenza A, Influenza B and SARS-CoV-2.

Multiplexing is a relevant strategy for biosensors to improve accuracy and decision-making due to the increased amount of simultaneously obtained information. Liposomes offer unique benefits for label-based multiplexing since a variety of different marker molecules can be encapsulated, leading to intrinsic signal amplification and enabling a variety of detection formats. We successfully developed an electrochemical (EC) liposome-based platform technology for the simultaneous detection of at least three analytes by studying parameters to ensure specific and sensitive bioassay performance. Influenza A and B and SARS-CoV-2 sequences served as model system in a standard sandwich hybridization assay. Studies included encapsulants, probe distribution on liposomes and capture beads, assay setup and interferences between liposomes to also ensure a generalization of the platform. Ruthenium hexamine(III), potassium hexacyanoferrate(II) and m-carboxy luminol, when encapsulated separately into a liposome, provided desirable long-term stability of at least 12 months and no cross-signals between liposomes. Through the optimization process, low limits of detections of 1.6 nmol L-1, 125 pmol L-1 and 130 pmol L-1, respectively, were achieved in a multiplexed assay setup, which were similar to singleplex assays. Non-specific interactions were limited to 25.1%, 7.6% and 7.5%, respectively, through sequential liposome incubations and singleplex capture bead designs. Here, ruthenium hexamine liposomes had only mediocre performance so that low overall signal strength translated into higher LODs and worse specificity. A different marker such as ferroin may be an option in the future. The identification of further electrochemical markers will provide new opportunities for liposomes to function as multiplex, orthogonal or internal standard labels in electrochemical bioassays.

Glomerulonefritis aguda posinfecciosa asociada al virus de la gripe B y enterovirus / Postinfectious acute glomerulonephritis associated to influenza B virus and enterovirus

La glomerulonefritis aguda posinfecciosa (GNAPI) es una lesión inflamatoria con afectación principal del glomérulo generada por una infección extrarrenal. Su patogenia es inmune, desencadenada por gran variedad de gérmenes: bacterias, virus y hongos. La causa más frecuente es la glomerulonefritis posestreptocócica (GNAPE). El caso que presentamos fue causado por gripe B y enterovirus, agentes etiológicos de presentación poco frecuente, con manifestación clínica similar a una glomerulonefritis posestreptocócica. Concluimos que, ante una clínica de síndrome nefrítico, se deben tener en cuenta los antecedentes víricos, para hacer un diagnóstico precoz (AU)

Acute post-infectious glomerulonephritis (APIGN) is an inflammatory lesion with main involvement of the glomerulus triggered by an extrarenal infection. Its pathogenesis is immune, triggered by a wide variety of germs: bacteria, viruses and fungi. The most common cause is poststreptococcal glomerulonephritis (PSAGN). The case that we present was associated to influenza B and enterovirus, etiological agents of infrequent presentation, with clinical manifestations similar to post-streptococcal glomerulonephritis. We conclude that, when faced with a nephritic syndrome clinic, the viral history should be taken into account to make an early diagnosis. (AU)

Circulation and seasonality of influenza viruses in different transmission zones in Africa.

BACKGROUND: Influenza is responsible for more than 5 million severe cases and 290,000 to 650,000 deaths every year worldwide. Developing countries account for 99% of influenza deaths in children under 5 years of age. This paper aimed to determine the dynamics of influenza viruses in African transmission areas to identify regional seasonality for appropriate decision-making and the development of regional preparedness and response strategies. METHODS: We used data from the WHO FluMart website collected by National Influenza Centers for seven transmission periods (2013-2019). We calculated weekly proportions of positive influenza cases and determined transmission trends in African countries to determine the seasonality. RESULTS: From 2013 to 2019, influenza A(H1N1)pdm2009, A(H3N2), and A(H5N1) viruses, as well as influenza B Victoria and Yamagata lineages, circulated in African regions. Influenza A(H1N1)pdm2009 and A(H3N2) highly circulated in northern and southern Africa regions. Influenza activity followed annual and regional variations. In the tropical zone, from eastern to western via the middle regions, influenza activities were marked by the predominance of influenza A subtypes despite the circulation of B lineages. One season was identified for both the southern and northern regions of Africa. In the eastern zone, four influenza seasons were differentiated, and three were differentiated in the western zone. CONCLUSION: Circulation dynamics determined five intense influenza activity zones in Africa. In the tropics, influenza virus circulation waves move from the east to the west, while alternative seasons have been identified in northern and southern temperate zones. Health authorities from countries with the same transmission zone, even in the absence of local data based on an established surveillance system, should implement concerted preparedness and control activities, such as vaccination.

Clinical validation of a multiplex PCR-based detection assay using saliva or nasopharyngeal samples for SARS-Cov-2, influenza A and B.

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.

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.

Severe influenza virus infection in children admitted to the PICU: Comparison of influenza A and influenza B virus infection.

Although the influenza virus usually causes a self-limiting disease, deaths are reported even in children without risk factors. We aimed to identify the clinical features, mortality associated with severe influenza A and B virus infections of children admitted to the pediatric intensive care unit (PICU). We conducted a retrospective study of children with confirmed influenza infection between 2012 and 2019 who were admitted to the PICU. Demographic features, risk factors, clinical data, microbiological data, complications, and outcomes were collected. Over seven influenza seasons (2012-2011 to 2015-2016), 713 children diagnosed with laboratory-confirmed influenza-related LRTI, and PICU admission was needed in 6% (46/713) of the patients. Thirty-one patients (67.4%) were diagnosed with influenza A and 15 patients were diagnosed with influenza B. Epidemiologic and clinical characteristics were similar in both influenza types, lactate dehydrogenase levels were significantly higher for influenza A than for influenza B infections. Although the influenza A to B ratio among the patients admitted to the PICU was 2.06, the percentage of cases requiring PICU admission was nearly two times higher in influenza B cases. There was no statistically significant difference in disease severity and complications in patients with influenza A and influenza B.

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.

The first epidemiological and virological influenza surveillance in the Republic of Guinea revealed the predominance of influenza A/H3N2 and B Victoria viruses.

Little is known about respiratory viruses infection in Guinea. Influenza surveillance has not been implemented in Guinea mainly because of the paucity of laboratory infrastructure and capacity. This paper presents the first influenza surveillance data in Guinea.Swabs were obtained from August 2018 through December 2019 at influenza sentinel sites and transported to the Institut National de Santé Publique for testing. Ribonucleic acid was extracted and tested for the presence of influenza A and B by real-time reverse transcription-polymerase chain reaction (RT-PCR). Positive samples were further characterised to determine the subtypes and lineages of influenza viruses.A total of 862 swabs were collected and tested. Twenty-three per cent of samples tested positive for influenza A and B viruses. Characterisation of positive specimens identified influenza A/H1N1pmd09 (2.5%), influenza A/H3N2 (57.3%), influenza B/Victoria lineage (36.7%) and 7 (3.5%) influenza B with undetermined lineage. Influenza B virus activity clustered in August through November while influenza A/H3N2 displayed two clusters of activities that appeared in May through August and November through December.For the first time in Guinea, the epidemiology, diversity and period of circulation of influenza viruses were studied. The results indicate the predominance and the periods of activities of influenza B Victoria lineage and influenza A/H3N2 which are important information for preventive strategies. It is warranted to extend the influenza surveillance to other parts of Guinea to better understand the epidemiology of the viruses and monitor the emergence of influenza strains with pandemic potential.

Exploring the epidemiological changes of common respiratory viruses since the COVID-19 pandemic: a hospital study in Hangzhou, China.

Adenovirus, respiratory syncytial virus, and influenza virus are common causes of respiratory infections. The COVID-19 pandemic had a significant impact on their prevalence. The aim of this study was to analyze the epidemic changes of common respiratory viruses in the Affiliated Hospital of Hangzhou Normal University in Hangzhou, China, from October of 2017 to February of 2021. We collected statistics from 121,529 patients in the outpatient and inpatient departments of the hospital who had throat or nose swabs collected for testing for four virus antigens by the colloidal gold method. Of these, 13,200 (10.86%) were positive for influenza A virus, 8,402 (6.91%) were positive for influenza B virus, 6,056 (4.98%) were positive for adenovirus, and 4,739 (3.90%) were positive for respiratory syncytial virus. The positivity rates of the influenza A virus (0-14 years old, P = 0.376; over 14 years old, P = 0.197) and respiratory syncytial virus (0-14 years old, P = 0.763; over 14 years old, P = 0.465) did not differ significantly by gender. After January of 2020, influenza virus infection decreased significantly. The positivity rate of respiratory syncytial virus remained high, and its epidemic season was similar to before. Strict respiratory protection and regulation of crowd activities have a great impact on the epidemic characteristics of viruses. After major changes in the public health environment, virus epidemics and their mutations should be monitored closely, extensively, and continuously.

Influenza B infection and Kawasaki disease in an adolescent during the COVID-19 pandemic: a case report. / Infecção por Influenza B e doença de Kawasaki em adolescente durante a pandemia da COVID-19: relato de caso.

We report a case of Influenza B infection and Kawasaki disease in an adolescent, diagnosed during the COVID-19 pandemic. An asthmatic female adolescent presented with fever and flu-like symptoms for 7 days and was admitted with acute respiratory failure requiring mechanical ventilation. She progressed with hemodynamic instability responsive to vasoactive drugs. Antibiotic therapy and support measures were introduced, showing progressive hemodynamics and respiratory improvement, however with persistent fever and increased inflammatory markers. During the hospitalization, she developed bilateral non-purulent conjunctivitis, hand and feet desquamation, strawberry tongue, and cervical adenopathy, and was diagnosed with Kawasaki disease. She was prescribed intravenous immunoglobulin and, due to the refractory clinical conditions, corticosteroid therapy was added; 24 hours later, the patient was afebrile. No coronary changes were found. A full viral panel including COVID-19 C-reactive protein and serology could only isolate the Influenza B virus. During the hospitalization, she was diagnosed with pulmonary thromboembolism; coagulopathies were investigated, and she was diagnosed with heterozygous factor V Leiden mutation. There is a potential association between Kawasaki disease and infection with Influenza B or with other viruses such as coronavirus. Therefore, this association should be considered in pediatric patients, adolescents included, with prolonged febrile conditions.

Apresentação de um caso de infecção por Influenza B e doença de Kawasaki em adolescente ocorrido durante a pandemia da COVID-19. Adolescente asmática evoluiu com febre e síndrome gripal por 7 dias e deu entrada com quadro de insuficiência respiratória aguda, necessitando de intubação orotraqueal. Evoluiu também com instabilidade hemodinâmica respondedora ao uso de droga vasoativa. Foram introduzidas antibioticoterapia e medidas de suporte. Apresentou melhora hemodinâmica e respiratória progressiva, porém mantinha febre e alteração de provas inflamatórias. Durante internação, evoluiu com conjuntivite não purulenta bilateral, descamação de mão e pés, língua em framboesa e linfonodomegalia cervical, recebendo diagnóstico de doença de Kawasaki. Recebeu gamaglobulina e, por conta de quadro clínico refratário, foi administrado também corticoide, evoluindo afebril 24 horas após. Não apresentou alterações coronarianas. O único agente isolado foi Influenza B, mesmo realizando painel viral e investigação para COVID-19 com reação em cadeia da polimerase e sorologia. Durante internação, apresentou tromboembolismo pulmonar, e, em investigação de coagulopatias, foi diagnosticada com mutação em heterozigose de fator V de Leiden. Há uma potencial associação entre doença de Kawasaki e infecção por Influenza B ou outros vírus, como o coronavírus e, por isso, esses diagnósticos devem ser investigados nos pacientes pediátricos, incluindo adolescentes, com quadros febris prolongados.

European multicenter evaluation of Xpert® Xpress SARS-CoV-2/Flu/RSV test.

Rapid diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are paramount for reducing the spread of the current pandemic. During additional seasonal epidemics with influenza A/B and respiratory syncytial virus (RSV), the clinical signs and symptoms cannot be distinguished easily from SARS-CoV-2. Therefore, a new assay combining four targets in the form of the new Xpert Xpress SARS-CoV-2/Flu/RSV assay was evaluated. The assay was compared to the Xpert Xpress SARS-CoV-2, Xpert Xpress Flu/RSV, Seegene Flu/RSV, influenza A/B r-gene® and RSV/hMPV r-gene®. A total of 295 nasopharyngeal and throat swabs were tested at four institutes throughout Europe including 72 samples positive for SARS-CoV-2, 65 for influenza A, 47 for influenza B, and 77 for RSV. The sensitivity of the new assay was above 95% for all targets, with the highest for SARS-CoV-2 (97.2%). The overall correlation of SARS-CoV-2 Ct values between Xpert Xpress SARS-CoV-2 assay and Xpert Xpress SARS-CoV-2/Flu/RSV assay was high. The agreement between Ct values above 30 showed the multiplex giving higher Ct values for SARS-CoV-2 on average than the singleplex assay. In conclusion, the new assay is a rapid and reliable alternative with less hands-on time for the detection of not one, but four upper respiratory tract pathogens that may circulate at the same time.