Epidemiology and genetic characterization of influenza viruses circulating in Bhutan in 2022.

INTRODUCTION: Influenza (Flu) causes considerable morbidity and mortality globally, and in Bhutan, Flu viruses are a leading cause of acute respiratory infection and cause outbreaks during Flu seasons. In this study, we aim to analyze the epidemiology and the genetic characterization of Flu viruses circulated in Bhutan in 2022. METHOD: Respiratory specimens were collected from patients who meet the case definition for influenza-like illness (ILI) and severe acute respiratory infection (SARI) from sentinel sites. Specimens were tested for Flu and SARS-CoV-2 viruses by RT-PCR using the Multiplex Assay. Selected positive specimens were utilized for Flu viral genome sequencing by next-generation sequencing. Descriptive analysis was performed on patient demographics to see the proportion of Flu-associated ILI and SARI. All data were analyzed using Epi Info7 and QGIS 3.16 software. RESULT: A weekly average of 16.2 ILI cases per 1000 outpatient visits and 18 SARI cases per 1000 admitted cases were reported in 2022. The median age among ILI was 12 years (IQR: 5-28) and SARI was 6.2 (IQR: 2.5-15) years. Flu A(H3N2) (70.2%) subtype was the most predominant circulating strain. Flu A(H1N1)pdm09 and Flu B viruses belonged to subclades that were mismatched to the vaccine strains recommended for the 2021-2022 season but matched the vaccine strain for the 2022-2023 season with vaccine efficacy 85.14% and 88.07% respectively. Flu A(H3N2) virus belonged to two subclades which differed from the vaccine strains recommended in both the 2021-2022 and 2022-2023 seasons with vaccine efficacy 68.28%. CONCLUSION: Flu virus positivity rates were substantially elevated during the Flu season in 2022 compared to 2021. Flu A(H3N2) subtype was the most predominant circulating strain in the country and globally. Genetic characterization of the Flu viruses in Bhutan showed a close relatedness of high vaccine efficacy with the vaccine strain that WHO recommended for the 2022-23 season.

Prevalence of Influenza B/Yamagata Viruses From Season 2012/2013 to 2021/2022 in Italy as an Indication of a Potential Lineage Extinction.

BACKGROUND: Influenza B/Yamagata viruses exhibited weak antigenic selection in recent years, reducing their prevalence over time and requiring no update of the vaccine component since 2015. To date, no B/Yamagata viruses have been isolated or sequenced since March 2020. METHODS: The antibody prevalence against the current B/Yamagata vaccine strain in Italy was investigated: For each influenza season from 2012/2013 to 2021/2022, 100 human serum samples were tested by haemagglutination inhibition (HAI) assay against the vaccine strain B/Phuket/3073/2013. In addition, the sequences of 156 B/Yamagata strains isolated during the influenza surveillance activities were selected for analysis of the haemagglutinin genome segment. RESULTS: About 61.9% of the human samples showed HAI antibodies, and 21.7% had protective antibody levels. The prevalence of antibodies at protective levels in the seasons between the isolation of the strain and its inclusion in the vaccine was between 11% and 25%, with no significant changes observed in subsequent years. A significant increase was observed in the 2020/2021 season, in line with the increase in influenza vaccine uptake during the pandemic. Sequence analysis showed that from 2014/2015 season onward, all B/Yamagata strains circulating in Italy were closely related to the B/Phuket/2013 vaccine strain, showing only limited amino acid variation. CONCLUSIONS: A consistent prevalence of antibodies to the current B/Yamagata vaccine strain in the general population was observed. The prolonged use of a well-matched influenza vaccine and a low antigenic diversity of B/Yamagata viruses may have facilitated a strong reduction in B/Yamagata circulation, potentially contributing to the disappearance of this lineage.

Detection of influenza virus in urban wastewater during the season 2022/2023 in Sicily, Italy.

Introduction: Seasonal influenza generally represents an underestimated public health problem with significant socioeconomic implications. Monitoring and detecting influenza epidemics are important tasks that require integrated strategies. Wastewater-based epidemiology (WBE) is an emerging field that uses wastewater data to monitor the spread of disease and assess the health of a community. It can represent an integrative surveillance tool for better understanding the epidemiology of influenza and prevention strategies in public health. Methods: We conducted a study that detected the presence of Influenza virus RNA using a wastewater-based approach. Samples were collected from five wastewater treatment plants in five different municipalities, serving a cumulative population of 555,673 Sicilian inhabitants in Italy. We used the RT-qPCR test to compare the combined weekly average of Influenza A and B viral RNA in wastewater samples with the average weekly incidence of Influenza-like illness (ILI) obtained from the Italian national Influenza surveillance system. We also compared the number of positive Influenza swabs with the viral RNA loads detected from wastewater. Our study investigated 189 wastewater samples. Results: Cumulative ILI cases substantially overlapped with the Influenza RNA load from wastewater samples. Influenza viral RNA trends in wastewater samples were similar to the rise of ILI cases in the population. Therefore, wastewater surveillance confirmed the co-circulation of Influenza A and B viruses during the season 2022/2023, with a similar trend to that reported for the weekly clinically confirmed cases. Conclusion: Wastewater-based epidemiology does not replace traditional epidemiological surveillance methods, such as laboratory testing of samples from infected individuals. However, it can be a valuable complement to obtaining additional information on the incidence of influenza in the population and preventing its spread.

Surveillance of influenza A and B viruses from community and hospital wastewater treatment plants.

Influenza virus is a well-known pathogen that can cause epidemics and pandemics. Several surveillance methods are being followed to monitor the transmission patterns and spread of influenza in the community. Wastewater-based Epidemiology (WBE) can serve as an additional tool to detect the presence of influenza viruses. The current study primarily focuses on surveillance of Influenza A and Influenza B in wastewater treatment plant (WWTP) samples. A total of 100 wastewater samples were collected in July (n = 50) and August (n = 50) 2023 from four different WWTPs in Manipal and Udupi, district of Karnataka, India. The WWTP samples were processed and tested by Real-Time reverse transcriptase PCR (RT-PCR). The data generated was analysed in comparison with the clinical Influenza cases. Of the 100 samples, 18 (18%) tested positive for Influenza A virus and 2 (2%) tested positive for Influenza B virus, with a viral load ranging 1.4 x 102-2.2 x 103 gc/L for influenza A virus and 5.2 x 103-7.7 x 103gc/L for influenza B virus. On correlating the WWTP positivity with clinical case, it was found that influenza clinical cases and virus positivity in wastewater increased simultaneously, emphasizing WBE as a concurrent method for monitoring influenza virus activity.

Point-of-Care Multiplex Detection of Respiratory Viruses.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

An end-point multiplex RT-PCR for SARS-CoV-2, Influenza A and B detection, including simultaneous RNAse P amplification: a timely tool for more accessible differential diagnosis.

The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.

Molecular epidemiology and phylogenetic analysis of influenza viruses A (H3N2) and B/Victoria during the COVID-19 pandemic in Guangdong, China.

BACKGROUND: Non-pharmaceutical measures and travel restrictions have halted the spread of coronavirus disease 2019 (COVID-19) and influenza. Nonetheless, with COVID-19 restrictions lifted, an unanticipated outbreak of the influenza B/Victoria virus in late 2021 and another influenza H3N2 outbreak in mid-2022 occurred in Guangdong, southern China. The mechanism underlying this phenomenon remains unknown. To better prepare for potential influenza outbreaks during COVID-19 pandemic, we studied the molecular epidemiology and phylogenetics of influenza A(H3N2) and B/Victoria that circulated during the COVID-19 pandemic in this region. METHODS: From January 1, 2018 to December 31, 2022, we collected throat swabs from 173,401 patients in Guangdong who had acute respiratory tract infections. Influenza viruses in the samples were tested using reverse transcription-polymerase chain reaction, followed by subtype identification and sequencing of hemagglutinin (HA) and neuraminidase (NA) genes. Phylogenetic and genetic diversity analyses were performed on both genes from 403 samples. A rigorous molecular clock was aligned with the phylogenetic tree to measure the rate of viral evolution and the root-to-tip distance within strains in different years was assessed using regression curve models to determine the correlation. RESULTS: During the early period of COVID-19 control, various influenza viruses were nearly undetectable in respiratory specimens. When control measures were relaxed in January 2020, the influenza infection rate peaked at 4.94% (39/789) in December 2021, with the influenza B/Victoria accounting for 87.18% (34/39) of the total influenza cases. Six months later, the influenza infection rate again increased and peaked at 11.34% (255/2248) in June 2022; influenza A/H3N2 accounted for 94.51% (241/255) of the total influenza cases in autumn 2022. The diverse geographic distribution of HA genes of B/Victoria and A/H3N2 had drastically reduced, and most strains originated from China. The rate of B/Victoria HA evolution (3.11 × 10-3, P < 0.05) was 1.7 times faster than before the COVID-19 outbreak (1.80 × 10-3, P < 0.05). Likewise, the H3N2 HA gene's evolution rate was 7.96 × 10-3 (P < 0.05), which is 2.1 times faster than the strains' pre-COVID-19 evolution rate (3.81 × 10-3, P < 0.05). CONCLUSIONS: Despite the extraordinarily low detection rate of influenza infection, concealed influenza transmission may occur between individuals during strict COVID-19 control. This ultimately leads to the accumulation of viral mutations and accelerated evolution of H3N2 and B/Victoria viruses. Monitoring the evolution of influenza may provide insights and alerts regarding potential epidemics in the future.

Analysis of the epidemic characteristics of common respiratory pathogens infection in children in a 3A special hospital.

OBJECTIVE: To analyze the epidemic characteristics of common respiratory tract infection pathogens in children with respiratory tract infection, and provide scientific basis for the prevention and control of respiratory tract infection. METHODS: A retrospective collection of clinical data was conducted on 11,538 children with respiratory tract infections at Luoyang Maternal and Child Health Hospital from December 2022 to November 2023. The types of respiratory tract infections, including upper and lower respiratory tract infections, as well as five respiratory pathogens: influenza A virus (influenza A), influenza B virus (influenza B virus, adenovirus (ADV), respiratory syncytial virus (RSV), and Mycoplasma pneumoniae (MP) infections, were analyzed and compared for different genders, ages, temperatures, and air quality in different months; And the changes of five pathogens in children with respiratory tract infections of different disease severity. RESULTS: From December 2022 to November 2023, a total of 11,538 children with respiratory infections were included in the analysis, including 6436 males and 5102 females, with an age of 4.92 ± 2.03 years. The proportion of upper respiratory tract infections is as high as 72.17%, and lower respiratory tract infections account for 27.83%. Among them, 2387 were positive for Flu A antigen, with a positive rate of 20.69%, 51 cases were positive for Flu B antigen, and the positive rate was 0.4%, 1296 cases were positive for adv antigen, with a positive rate of 11.23%, 868 cases were positive for RSV antigen, with a positive rate of 7.52%, 2481 cases were positive for MP IgM antibody or MP antigen, and the positive rate was 21.50%. Flu B in male children The infection rate of ADV and MP was higher than that of female children (p < 0.05); Among children in different age groups, the older the age, the older the Flu A The higher the infection rate of MP (p < 0.05), the higher the positive rate of RSV in children with younger age (p < 0.05). The positive rate of ADV in children aged 3-6 years and > 6 years was higher than that in children aged 0-3 years (p < 0.05); Flu A and MP are popular throughout the year, and the positive rate peaks during the period of temperature rise and air quality decline from February to March, and during the period of temperature drop and air quality index rise from August to November, The positive rate of RSV peaked after the turning point of temperature rise from March to April. The infection rate was higher during the period of sharp decline in air quality from March to May and sharp decline in temperature in November, The positive rate of ADV was higher at the turning point of temperature rise from February to March, and then the infection rate decreased. During the period of sharp temperature drop from August to November, the positive rate increased sharply, and the peak of infection occurred; As the disease worsens, The positive rates of Flu A, Flu B, RSV, MP and combined infection with more than two pathogens were all increased (p < 0.05). CONCLUSION: After the new coronavirus epidemic in 2022, Flu A and MP have the highest infection rate of respiratory pathogens in children, showing a peak growth in general, with epidemic characteristics changing with environmental temperature, air quality and seasons. The main disease type is upper respiratory tract infection, MP and adv infections were mainly in male children, Flu A, MP and ADV infections are more common in older children, RSV infection was more common in younger children; Flu A, Flu B, RSV and MP infection and the co infection of more than two pathogens may more easily lead to the occurrence of severe pneumonia.

Prevalence of Co-Infections in Primary Care Patients with Medically Attended Acute Respiratory Infection in the 2022/2023 Season.

In the post-pandemic period, an endemic circulation of respiratory viruses has been re-established. Respiratory viruses are co-circulating with SARS-CoV-2. We performed a retrospective analysis of co-infections in primary care patients with medically attended acute respiratory infections (MAARI) who consulted from week 40/2022 to week 39/2023 and were tested for a panel of respiratory viruses. Out of 2099 samples tested, 1260 (60.0%) were positive for one virus. In 340 samples, co-infection was detected: two viruses in 281 (13.4%), three viruses in 51 (2.4%), and four viruses in eight (0.4%) samples. Respiratory viruses co-infected the patients with MAARI at very different rates. The lowest rates of co-infections were confirmed for influenza B (13.8%) and influenza A (22.9%) and the highest for human bocaviruses (84.0%) and human parechoviruses (82.1%). Co-infections were detected in 28.2% of SARS-CoV-2 positive samples. SARS-CoV-2 has never been co-infected with influenza B virus, enterovirus or adenovirus, although the latter was found as a co-infecting virus with all other respiratory viruses tested. The rate of co-infections decreased significantly with increasing age (p-value 0.000), and no difference was found regarding gender (p-value 0.672). It is important to understand the epidemiology of respiratory co-infections for prevention and management decisions in patients with MAARI.

Clinical Performance of the BD Respiratory Viral Panel for BD MAX™ System in Detecting SARS-CoV-2, Influenza A and B, and Respiratory Syncytial Virus.

Using a nasopharyngeal (NP) or anterior nasal (NS) swab from prospectively collected or retrospective specimens, we assessed the clinical performance of the BD Respiratory Viral Panel (BD RVP) for BD MAX System against FDA-cleared or authorized comparators. Across prospective and retrospective specimens, positive percent agreement (PPA) was ≥ 98.4% for SARS-CoV-2, ≥ 96.7% for influenza (flu) A, ≥ 91.7% for respiratory syncytial virus (RSV), and 100% for flu B (retrospective only) while negative percent agreement (NPA) was ≥ 97.7% across all targets, leading to the assay FDA clearance. A head-to-head comparison of NS versus NP results with BD RVP was also performed; PPA was ≥ 90% and NPA ≥ 98.2% for SARS-CoV-2, flu A and RSV. These findings confirm that the BD MAX RVP assay performs well for detection and differentiation of the three viruses in NP and NS specimens, with strong interrater agreements for NS versus NP comparisons.

[Analysis of epidemiological characteristics of respiratory pathogens in children with influenza-like illnesses in a children's hospital in Beijing from 2022 to 2023].

To investigate the status and epidemiological characteristics of respiratory pathogens infections in children with influenza-like illnesses (ILI) in Beijing Children's Hospital from 2022 to 2023. A dual amplification technique was used to detect nucleic acids of seven common respiratory pathogens, including influenza A virus (Flu A), influenza B virus (Flu B), mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus (ADV), and Chlamydia pneumoniae (CP), in outpatient and inpatient children (aged 0-18 years) with influenza-like symptoms who sought medical care at Beijing Children's Hospital, from January 2022 to March 2023. A total of 43 663 children were included in the study, of which 27 903 tested positive for respiratory pathogens with a total detection rate of 63.91%. Flu A had the highest detection rate of 69.93% (27 332/39 084), followed by MP about 13.22% (380/2 875). The total detection rate of RSV, PIV and ADV was 7.69% (131/1 704). Flu B had a detection rate of 0.16% (64/39 084). No CP was detected in this study. A total of 7 cases of dual infections were detected, with a detection rate of 0.41% (7/1 704). The Chi-square test was used to analyze the differences in detection rates of pathogens among different genders, age groups, and different seasons. Among the seven pathogens, only Flu A had statistically significant differences in gender (χ2=16.712, P<0.001). The detection rates of Flu A and MP showed an increasing trend with age (both P trend<0.001), while the detection rates of RSV and PIV showed a decreasing trend with age (both P trend<0.001). Flu A had its epidemic peak in winter and spring, with detection rates of 61.30% (3 907/6 374) and 77.47% (23 207/29 958) respectively; MP and PIV had higher detection rates in autumn (25.14% and 7.64% respectively); RSV showed a relatively higher detection rate in winter (8.69%); Flu B and ADV had lower detection rates throughout the study period (0.16% and 1.17% respectively). In conclusion, children with ILI in 2022-2023 were mainly infected with a single respiratory pathogen, and occasionally dual pathogen infections were observed. Among them, the detection rate of Flu A was the highest, and only Flu A showed a gender difference in detection rate. As the age of the children patients increased, the detection rate of Flu A and MP showed an increasing trend, while RSV and PIV showed a decreasing trend. The prevalence of Flu A, Flu B, MP, PIV, and RSV were seasonal.

One-step time-resolved cascade logic gate microfluidic chip for home testing of SARS-CoV-2 and flu B.

Home testing technology strategy is critical for early screening of disease. However, current home testing technologies often require complex processes, which limits their application. In this study, a time-resolved cascade logic gate microfluidic chip (TCLMC) was revealed to enable capillary force-based one-step operation without manual intervention or professional equipment. By analogy with logic gates in the circuit, TCLMC could automatically control the fluid flow and regulate the incubation time to optimize the immunoassay. The limit of detection of TCLMC for SARS-CoV-2 and influenza B virus (Flu B) was 134.94 and 79.17 pg mL-1 within 10 min. Additionally, this study tested saliva samples from 12 Flu B patients and 24 healthy controls to verify its clinical application. The results showed that TCLMC had high sensitivity (100%), specificity (100%), and accuracy (100%). This study provides a new one-step strategy for home testing and demonstrates its great potential in the diagnosis field.

Diagnostic accuracy of Savanna RVP4 (QuidelOrtho) for the detection of Influenza A virus, RSV, and SARS-CoV-2.

Seasonal increase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV) require rapid diagnostic test methods for the management of respiratory tract infections. In this study, we compared the diagnostic accuracy of Savanna RVP4 (RVP4, QuidelOrtho) with Xpert Xpress Plus SARS-CoV-2/Flu/RSV (Xpert, Cepheid). Nasopharyngeal swabs from patients treated at a tertiary care hospital (Germany) were tested for SARS-CoV-2, Flu A/B, and RSV by RVP4 to assess diagnostic accuracy (reference standard: Xpert). The intra and inter assay precision of Ct-values was assessed by repeated test in triplicates (on day 1) and duplicates (days 2-3). All patients with a physician's order for a multiplex test for SARS-CoV-2, Flu, and RSV test were included. Duplicate swabs from the same patient, samples with a total volume ≤1 mL, or inappropriate shipment/storage were excluded. In total, 229 swabs were included between September 2023 and February 2024. The concordance between both tests was 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.6% (RSV). Flu B was not detected by both tests. The RVP4 test had a sensitivity of 85%-95% and a specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV. The intra and inter assay precision of Ct-values from RVP4 was 3% and 2% (SARS-CoV-2), 5% and 4% (Flu A), and 0% and 3% (RSV), respectively. The Savanna RVP4 has a favorable diagnostic accuracy for the detection of SARS-CoV-2, Flu A, and RSV. IMPORTANCE: We assessed the diagnostic accuracy of a new point-of-care test for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV). The new test has a concordance with the reference standard of 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.1% (RSV). The sensitivity of 85%-95% and specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV is comparable with similar nucleic acid amplification-based point of care tests but at lower costs.

Modified transport medium for improving influenza virus detection.

Background: Accurate detection of influenza virus in clinical samples requires correct execution of all aspects of the detection test. If the viral load in a sample is below the detection limit, a false negative result may be obtained. To overcome this issue, we developed a modified transport medium (MTM) for clinical sample transportation to increase viral detection sensitivity. Method: We first validated the MTM using laboratory-stocked influenza A viruses (IAVs: H1N1, H3N2, H7N3, H9N2) and influenza B viruses (IBVs: Yamagata, Victoria). We also tested clinical samples. A total of 110 patients were enrolled and a pair of samples were collected to determine the sensitivity of real-time polymerase chain reaction (RT-PCR) following MTM treatment. Result: After 24 h culturing in MTM, the viral loads were increased, represented by a 10-fold increase in detection sensitivity for H1N1, H9N2, and IBVs, a 100-fold increase for H3N2, and a 1,000-fold increase for H7N3. We further tested the effects of MTM on 19 IAV and 11 IBV stored clinical samples. The RT-PCR results showed that the positive detection rate of IAV samples increased from 63.16% (12/19) without MTM culturing to 78.95% (15/19) after 48 h culturing, and finally 89.47% (17/19) after 72 h culturing. MTM treatment of IBV clinical samples also increased the positive detection rate from 36.36% (4/11, 0 h) to 63.64% (7/11, 48 h) to 72.73% (8/11, 72 h). For clinical samples detected by RT-PCR, MTM outperformed other transport mediums in terms of viral detection rate (11.81% increase, P=0.007). Conclusion: Our results demonstrated that the use of MTM for clinical applications can increase detection sensitivity, thus facilitating the accurate diagnosis of influenza infection.

Surveillance of respiratory viruses by aerosol screening in indoor air as an early warning system for epidemics.

The development of effective methods for the surveillance of seasonal respiratory viruses is required for the timely management of outbreaks. We aimed to survey Influenza-A, Influenza-B, RSV-A, Rhinovirus and SARS-CoV-2 surveillance in a tertiary hospital and a campus over 5 months. The effectiveness of air screening as an early warning system for respiratory viruses was evaluated in correlation with respiratory tract panel test results. The overall viral positivity was higher on the campus than in the hospital (55.0% vs. 38.0%). Influenza A was the most prevalent pathogen in both locations. There were two influenza peaks (42nd and 49th weeks) in the hospital air, and a delayed peak was detected on campus in the 1st-week of January. Panel tests indicated a high rate of Influenza A in late December. RSV-A-positivity was higher on the campus than the hospital (21.6% vs. 7.4%). Moreover, we detected two RSV-A peaks in the campus air (48th and 51st weeks) but only one peak in the hospital and panel tests (week 49). Although rhinovirus was the most common pathogen in panel tests, rhinovirus positivity was low in air samples. The air screening for Influenza-B and SARS-Cov-2 revealed comparable positivity rates with panel tests. Air screening can be integrated into surveillance programs to support infection control programs for potential epidemics of respiratory virus infections except for rhinoviruses.

Resurgence of influenza with increased genetic diversity of circulating viruses during the 2022-2023 season.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.

Clinical Evaluation of the Accuracy of the Panbio™ COVID-19/Flu A&B Rapid Panel: A Combination Antigen Rapid Diagnostic Test for the Omicron Variant and Influenza A Virus.

It is difficult to differentiate between coronavirus disease 2019 (COVID-19) and influenza based on the symptoms. In the present study, a newly developed antigen rapid diagnostic test (Ag-RDT) called Panbio™ COVID-19/Flu A&B that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B virus was evaluated. Its accuracy was evaluated using 235 pairs of nasopharyngeal samples collected from patients with respiratory symptoms and fever (>37.5°C). Reverse transcription polymerase chain reaction was used as a reference method to evaluate the accuracy of the SARS-CoV-2 detection. We confirmed the accuracy of the developed Ag-RDT against the Omicron variant where the sensitivity and specificity were 94.8% and 100%, respectively. In addition, to identify the influenza A virus, a noninferiority test was conducted using a commercial Ag-RDT, which has a sensitivity and specificity in comparison with viral culture of 94.8% and 98.4%, respectively. The positive and negative predictive values for influenza A virus were 98.5% and 98.1%, respectively, for the Panbio COVID-19/Flu A&B test. The evaluation of this newly developed Ag-RDT using clinical samples suggests that it has a high efficacy in clinical settings.

Epidemiological and Genetic Characteristics of Respiratory Viral Coinfections with Different Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Resurgence of seasonal influenza driven by A/H3N2 and B/Victoria in succession during the 2023-2024 season in Beijing showing increased population susceptibility.

During the COVID-19 pandemic, non-pharmaceutical interventions were introduced to reduce exposure to respiratory viruses. However, these measures may have led to an "immunity debt" that could make the population more vulnerable. The goal of this study was to examine the transmission dynamics of seasonal influenza in the years 2023-2024. Respiratory samples from patients with influenza-like illness were collected and tested for influenza A and B viruses. The electronic medical records of index cases from October 2023 to March 2024 were analyzed to determine their clinical and epidemiological characteristics. A total of 48984 positive cases were detected, with a pooled prevalence of 46.9% (95% CI 46.3-47.5). This season saw bimodal peaks of influenza activity, with influenza A peaked in week 48, 2023, and influenza B peaked in week 1, 2024. The pooled positive rates were 28.6% (95% CI 55.4-59.6) and 18.3% (95% CI 18.0-18.7) for influenza A and B viruses, respectively. The median values of instantaneous reproduction number were 5.5 (IQR 3.0-6.7) and 4.6 (IQR 2.4-5.5), respectively. The hospitalization rate for influenza A virus (2.2%, 95% CI 2.0-2.5) was significantly higher than that of influenza B virus (1.1%, 95% CI 0.9-1.4). Among the 17 clinical symptoms studied, odds ratios of 15 symptoms were below 1 when comparing influenza A and B positive inpatients, with headache, weakness, and myalgia showing significant differences. This study provides an overview of influenza dynamics and clinical symptoms, highlighting the importance for individuals to receive an annual influenza vaccine.