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.

Performance evaluation of the Panbio COVID-19/Flu A&B Panel for detection of SARS-CoV-2, influenza A, and influenza B antigens using mid-turbinate nasal swabs.

The Panbio COVID-19/Flu A&B Panel (Abbott) is an in vitro diagnostic rapid test designed for the qualitative detection of nucleocapsid proteins SARS-CoV-2 and nucleoprotein influenza A and B antigens in nasal mid-turbinate (NMT) swab specimens from symptomatic individuals meeting COVID-19 and influenza clinical and/or epidemiological criteria. This study, the largest global one to date using fresh samples, aimed to assess the diagnostic sensitivity and specificity of the Panbio COVID-19/Flu A&B Panel in freshly collected NMT swab specimens from individuals suspected of respiratory viral infection consistent with COVID-19 and/or influenza within the first 5 days of symptom onset compared with results obtained with the cobas SARS-CoV-2 and influenza A/B qualitative assay (cobas 6800/8800 systems), which were tested using nasopharyngeal swab samples. A total of 512 evaluable subjects were enrolled in the COVID-19 cohort across 18 sites, and 1,148 evaluable subjects were enrolled in the influenza cohort across 22 sites in the Asia-Pacific, Europe, and the USA. The Panbio COVID-19/Flu A&B Panel demonstrated a sensitivity of 80.4% and a specificity of 99.7% for COVID-19. For influenza A, the sensitivity and specificity rates were 80.6% and 99.3%, respectively. Likewise, for influenza B, the sensitivity and specificity rates were 80.8% and 99.4%, respectively. In conclusion, the Panbio COVID-19/Flu A&B Panel emerges as a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.4% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B. IMPORTANCE: The Panbio COVID-19/Flu A&B Panel is a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.0% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B.

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.

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.

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.

Establishment of a Raman nanosphere based immunochromatographic system for the combined detection of influenza A and B viruses' antigens on a single T-line.

A simple and rapid system based on Raman nanosphere (R-Sphere) immunochromatography was developed in this study for the simultaneous detection of Influenza A, B virus antigens on a single test line (T-line). Two types of R-Sphere with different characteristic Raman spectrum were used as the signal source, which were labeled with monoclonal antibodies against FluA, FluB (tracer antibodies), respectively. A mixture of antibodies containing anti-FluA monoclonal antibody and anti-FluB monoclonal antibody (capture antibody) was sprayed on a single T-line and goat anti-chicken IgY antibody was coated as a C-line, and the antigen solution with known concentration was detected by the strip of lateral flow immunochromatography based on surface-enhanced Raman spectroscopy (SERS). The T-line was scanned with a Raman spectrometer and SERS signals were collected. Simultaneous specific recognition and detection of FluA and FluB were achieved on a single T-line by analyzing the SERS signals. The findings indicated that the test system could identify FluA and FluB in a qualitative manner in just 15 minutes, with a minimum detection threshold of 0.25 ng ml-1, excellent consistency, and specificity. There was no interference with the other four respiratory pathogens, and it exhibited 8 times greater sensitivity compared to the colloidal gold test strip method. The assay system is rapid, sensitive, and does not require repetitive sample pretreatment steps and two viruses can be detected simultaneously on a single T-line by titrating one sample, which improves detection efficiency, and provide a reference for developing multiplexed detection techniques for other respiratory viruses.

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.

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.

Severe necrotizing tracheobronchitis caused by influenza B and methicillin-resistant Staphylococcus aureus co-infection in an immunocompetent patient.

PURPOSE AND METHOD: Necrotizing tracheobronchitis is a rare clinical entity presented as a necrotic inflammation involving the mainstem trachea and distal bronchi. We reported a case of severe necrotizing tracheobronchitis caused by influenza B and methicillin-resistant Staphylococcus aureus (MRSA) co-infection in an immunocompetent patient. CASE PRESENTATION: We described a 36-year-old man with initial symptoms of cough, rigors, muscle soreness and fever. His status rapidly deteriorated two days later and he was intubated. Bronchoscopy demonstrated severe necrotizing tracheobronchitis, and CT imaging demonstrated multiple patchy and cavitation formation in both lungs. Next-generation sequencing (NGS) and bronchoalveolar lavage fluid (BALF) culture supported the co-infection of influenza B and MRSA. We also found T lymphocyte and NK lymphocyte functions were extremely suppressed during illness exacerbation. The patient was treated with antivirals and antibiotics including vancomycin. Subsequent bronchoscopy and CT scans revealed significant improvement of the airway and pulmonary lesions, and the lymphocyte functions were restored. Finally, this patient was discharged successfully. CONCLUSION: Necrotizing tracheobronchitis should be suspected in patients with rapid deterioration after influenza B infection. The timely diagnosis of co-infection and accurate antibiotics are important to effective treatment.

Effectiveness of influenza vaccines in children aged 6 to 59 months: a test-negative case-control study at primary care and hospital level, Spain 2023/24.

During 2023/24, all children aged 6 to 59 months were targeted for seasonal influenza vaccination in Spain nationally. Using a test-negative case-control design with sentinel surveillance data, we estimated adjusted influenza vaccine effectiveness (IVE) against any influenza type to be 70% (95% confidence interval (CI): 51 to 81%) for primary care patients with acute respiratory illness (ARI) and 77% (95% CI: 21 to 93%) for hospitalised patients with severe ARI. In primary care, where most subtyped viruses (61%; 145/237) were A(H1N1), adjusted IVE was 77% (95% CI: 56 to 88%) against A(H1N1)pdm09.

Co-circulation of seasonal influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage viruses with further genetic diversification, EU/EEA, 2022/23 influenza season.

BackgroundInfluenza viruses can cause large seasonal epidemics with high healthcare impact and severity as they continually change their virological properties such as genetic makeup over time.AimWe aimed to monitor the characteristics of circulating influenza viruses over the 2022/23 influenza season in the EU/EEA countries. In addition, we wanted to compare how closely the circulating viruses resemble the viral components selected for seasonal influenza vaccines, and whether the circulating viruses had acquired resistance to commonly used antiviral drugs.MethodsWe performed a descriptive analysis of the influenza virus detections and characterisations reported by National Influenza Centres (NIC) from the 30 EU/EEA countries from week 40/2022 to week 39/2023 to The European Surveillance System (TESSy) as part of the Global Influenza Surveillance and Response System (GISRS).ResultsIn the EU/EEA countries, the 2022/23 influenza season was characterised by co-circulation of A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses. The genetic evolution of these viruses continued and clade 6B.1A.5a.2a of A(H1N1)pdm09, 3C.2a1b.2a.2b of A(H3N2) and V1A.3a.2 of B/Victoria viruses dominated. Influenza B/Yamagata-lineage viruses were not reported.DiscussionThe World Health Organization (WHO) vaccine composition recommendation for the northern hemisphere 2023/24 season reflects the European virus evolution, with a change of the A(H1N1)pdm09 component, while keeping the A(H3N2) and B/Victoria-lineage components unchanged.

Multiplex CLIA-waived point-of-care tests for SARS-CoV-2, influenza A and B, ± other respiratory pathogens: A systematic review.

BACKGROUND: Many multiplex CLIA-waived point of care tests are available in the United States for community pharmacists to incorporate into their practice. OBJECTIVE: To characterize the eleven multiplex CLIA-waived point of care tests available for detecting SARS-CoV-2 and influenza A and B ± other respiratory pathogens and summarize their performance characteristics from published studies. METHODS: Food and Drug Administration websites and databases were utilized to identify currently available tests. Literature searches were conducted and most recently updated through July 21, 2023 to identify studies examining the tests' performance. Primary literature was included if it reported positive and negative percent agreement (PPA and NPA, respectively) for SARS-CoV-2 or influenza A or B, had a comparator, and was conducted in a CLIA-waived environment. Relevant study characteristics, including number of samples, sample collection method, age of participants, comparator, PPA/NPA for SARS-CoV-2, influenza A and B, and respiratory syncytial virus (RSV) were extracted. Data extraction was performed by two authors with any discordant data reconciled by a third author. RESULTS: Twenty-eight studies met inclusion criteria. The PPA for SARS-CoV-2 ranged from 86.7% to 100%, and NPA ranged from 90.4% to 100%. The PPA for influenza A and B ranged from 92.9% to 100% and 95.3% to 100%, respectively, while NPA ranged from 96.6% to 100% and 98.2% to 100%, respectively. Both the Xpert Xpress Plus and SpotFire tests showed similar results for RSV with PPA ranging from 96.3% to 100% and NPA ranging from 98.4% to 100%. CONCLUSION: Currently available multiplex POCT for SARS CoV-2 and influenza A and B ± RSV have demonstrated efficacy in detecting multiple pathogens in a single test. Pathogen detection, costs, materials needed to perform the test (eg, readers), and study data (PPA and NPA) should be utilized to help determine which tests should be ordered for a particular practice setting.

[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.

The active form of the influenza cap-snatching endonuclease inhibitor baloxavir marboxil is a tight binding inhibitor.

Baloxavir marboxil (BXM) is an FDA-approved antiviral prodrug for the treatment of influenza A and B infection and postexposure prophylaxis. The active form, baloxavir acid (BXA), targets the cap-snatching endonuclease (PA) of the influenza virus polymerase complex. The nuclease activity delivers the primer for transcription, and previous reports have shown that BXA blocks the nuclease activity with high potency. However, biochemical studies on the mechanism of action are lacking. Structural data have shown that BXA chelates the two divalent metal ions at the active site, like inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase or ribonuclease (RNase) H. Here we studied the mechanisms underlying the high potency of BXA and how the I38T mutation confers resistance to the drug. Enzyme kinetics with the recombinant heterotrimeric enzyme (FluB-ht) revealed characteristics of a tight binding inhibitor. The apparent inhibitor constant (Kiapp) is 12 nM, while the I38T mutation increased Kiapp by ∼18-fold. Order-of-addition experiments show that a preformed complex of FluB-ht, Mg2+ ions and BXA is required to observe inhibition, which is consistent with active site binding. Conversely, a preformed complex of FluB-ht and RNA substrate prevents BXA from accessing the active site. Unlike integrase inhibitors that interact with the DNA substrate, BXA behaves like RNase H inhibitors that compete with the nucleic acid at the active site. The collective data support the conclusion that BXA is a tight binding inhibitor and the I38T mutation diminishes these properties.

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.

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.

Acute Myocardial Infarction after Laboratory-Confirmed Influenza Infection.

BACKGROUND: Acute myocardial infarction can be triggered by acute respiratory infections. Previous studies have suggested an association between influenza and acute myocardial infarction, but those studies used nonspecific measures of influenza infection or study designs that were susceptible to bias. We evaluated the association between laboratory-confirmed influenza infection and acute myocardial infarction. METHODS: We used the self-controlled case-series design to evaluate the association between laboratory-confirmed influenza infection and hospitalization for acute myocardial infarction. We used various high-specificity laboratory methods to confirm influenza infection in respiratory specimens, and we ascertained hospitalization for acute myocardial infarction from administrative data. We defined the "risk interval" as the first 7 days after respiratory specimen collection and the "control interval" as 1 year before and 1 year after the risk interval. RESULTS: We identified 364 hospitalizations for acute myocardial infarction that occurred within 1 year before and 1 year after a positive test result for influenza. Of these, 20 (20.0 admissions per week) occurred during the risk interval and 344 (3.3 admissions per week) occurred during the control interval. The incidence ratio of an admission for acute myocardial infarction during the risk interval as compared with the control interval was 6.05 (95% confidence interval [CI], 3.86 to 9.50). No increased incidence was observed after day 7. Incidence ratios for acute myocardial infarction within 7 days after detection of influenza B, influenza A, respiratory syncytial virus, and other viruses were 10.11 (95% CI, 4.37 to 23.38), 5.17 (95% CI, 3.02 to 8.84), 3.51 (95% CI, 1.11 to 11.12), and 2.77 (95% CI, 1.23 to 6.24), respectively. CONCLUSIONS: We found a significant association between respiratory infections, especially influenza, and acute myocardial infarction. (Funded by the Canadian Institutes of Health Research and others.).

Xpert Xpress Flu/RSV: Validation and impact evaluation at a large UK hospital trust.

Despite vaccination programs and antivirals, influenza remains a prominent cause of morbidity and mortality. The Xpert Xpress Flu/respiratory syncytial virus (RSV) test is a leading influenza point-of-care test, but its evaluation has been limited to nasopharyngeal samples. In addition, the clinical impacts of Xpress Flu/RSV have not yet been quantified. We evaluated the performance of Xpress Flu/RSV at three locations in a UK Hospital Trust against an existing laboratory assay. Multiple upper respiratory tract sample types were included. In addition, we calculated time saved by Xpert, and the associations between Xpert use and rates of early patient isolation and antiviral prescription as recorded at the time of the laboratory result being telephoned out. A total of 642 patients were included in the diagnostic performance analysis. There were 177 laboratory-confirmed cases of influenza A, 7 influenza B and 86 RSV. For influenza A, sensitivity and specificity were 96.6% (95% confidence interval [CI]: 92.8%-98.8%) and 98.1% (CI: 96.4%-99.1%), respectively. This was sustained across all locations and sample types. The negative predictive value was 98.7% (CI: 97.2%-99.4%). The median amount of time saved was 27.1 h. Xpert use was associated with sixfold higher rates of isolation and threefold higher rates of antiviral prescribing by the time the laboratory result was available. Sensitivity for RSV was lower at 86.0% (95% CI: 76.9%-92.6%). Xpert Xpress Flu/RSV reliably detects influenza A infection and has significant clinical impacts. Cartridge optimization is required to enable accurate multiplexing, including from a range of sample types.

Outcome predictors of influenza for hospitalization and mortality in children.

Severity of disease caused by influenza virus and the influencing factors that may be different. Moreover, the disease course actually may not be determined specifically in children because of lower seroprotection rates of children. Herein, the results clinic and outcome data of children with influenza from Turkey were reported. We present here the results from 2013 to 2017. Nasopharyngeal swab samples of the children with influenza were investigated via multiplex polymerase chain reaction. A total of 348 children were diagnosed with influenza; 143 (41.1%) were influenza A, 85 (24.4%) were influenza B, and 120 (34.5%) were mixt infection with other respiratory viruses. Fifty-four percent of children admitted to intensive care unit (ICU) were under 2 years of age (p = .001). Having an underlying disease was detected as the main predictor for both hospitalization and ICU stay according to multiple logistic regression analysis (odds ratio [OR], 11.784: 95% confidence interval [CI], 5.212-26.643; p = .001 and OR, 4.972: 95% CI, 2.331-10.605; p = .001, respectively). Neurological symptoms most frequently seen in cases who died (44.4%; p = .02). Lymphopenia was relatively higher (55.6%) and thrombocytopenia was most frequently seen in cases who died (77.8%) with a significant ratio (p = .001). Underlying diseases was found a risk factor for influenza being hospitalized and being admitted to ICU. Children under 2 years of age and with underlying diseases should be vaccinated particularly in countries where the influenza vaccination is still not routinely implemented in the immunization schedule. Highlights Underlying diseases is a risk factor for influenza to be hospitalized and admitted to ICU. Influenza vaccination is of great importance to prevent life-threatening complications of influenza, particularly in children require ICU admission. The possibility to reduce the outpatient visit number by vaccination has a great impact on disease burden in addition to the underestimated crucial social benefits, as well.

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.