Resurgence of respiratory syncytial virus infection during COVID-19 pandemic in Pune, India.

INTRODUCTION: Respiratory Syncytial Virus (RSV) is a leading cause of acute lower respiratory infection in children worldwide. Understanding its prevalence, variations, and characteristics is vital, particularly in the context of the COVID-19 pandemic. OBJECTIVE: The study aimed to investigate the RSV positivity rate, subtype prevalence, age and gender distribution, symptomatology, and co-infection rates during pre-pandemic and pandemic periods. METHODS: We analyzed data from 15,381 patients tested for RSV between 2017 and 2023. RESULTS: Our analysis revealed a 7.2% average RSV positivity rate in the pre-pandemic period, with significant fluctuations during the pandemic (1.5% in 2020 to 32.0% in 2021). We observed variations in RSVA and RSVB detection rates. The 0-4 years' age group was consistently the most affected, with a slight male predominance. Fever and cough were common symptoms. Therapeutic interventions, particularly antiviral usage and ventilation requirements, decreased during the pandemic. We also identified variations in co-infection rates with other respiratory viruses. CONCLUSION: Our study offers critical insights into the impact of the COVID-19 pandemic on RSV prevalence, subtype distribution, patient characteristics, and clinical management. These findings underscore the need for ongoing surveillance and adaptive public health responses.

The Burden of Respiratory Syncytial Virus in Children Under 2 Years of Age in a Rural Community in Maharashtra, India.

BACKGROUND: Globally, respiratory syncytial virus (RSV) is a common cause of acute lower tract infection (LRTI) in children younger than 2 years of age, but there are scant population-based studies on the burden of RSV illness in rural communities and no community studies in preterm infants. METHODS: Active surveillance of LRTI was performed in the community and hospital setting for the population of 93 tribal villages in Melghat, Central India, over 4 respiratory seasons. A nasopharyngeal swab was obtained from cases presenting as a severe LRTI for molecular analysis of respiratory pathogens including RSVA and B. RESULTS: High rates of RSV-associated LRTI were found in preterm and term infants beyond 6 months of age, extending into the second year of life. Community severe RSV LRTI rates for 0-11 months of age was 22.4 (18.6-27.0)/1000 child-years (CY) and the hospital-associated rate was 14.1 (11.1-17.8)/1000 CY. For preterm infants, these rates were 26.2 (17.8-38.5)/1000 CY and 12.6 (7.2-22.0)/1000 CY. Comparable rates in the first 6 months were 15.9 (11.8-21.4)/1000 CY and 12.9 (9.3-18.0)/1000 CY in term infants and 26.3 (15.4-45.0)/1000 CY and 10.1 (4.2-24.2)/1000 CY for preterms. The single RSV B season had higher incidences of RSV LRTI in every age group than the 2 RSV A seasons in both preterm and term infants. There were 11 deaths, all term infants. CONCLUSIONS: Studies restricted to the healthcare settings significantly underestimate the burden of RSV LRTI and preterm and term infants have comparable burdens of disease in this rural community.

Mortality From Respiratory Syncytial Virus in Children Under 2 Years of Age: A Prospective Community Cohort Study in Rural Maharashtra, India.

BACKGROUND: Although respiratory syncytial virus (RSV) is the most important viral cause of lower respiratory tract infection deaths in infants, there are few data on infant community deaths caused by RSV. METHODS: This was an active surveillance of children younger than 2 years of age in 93 villages, 5 primary health centers, and 3 hospitals serving these villages. Village health workers and counselors at the health facilities monitored all lower respiratory tract infections (LRTIs) in consented subjects. Children with severe, or very severe LRTIs and all who died, had nasopharyngeal swabs collected for detection of RSV by molecular methods. RESULTS: In the 12 134 subjects, there were 2064 episodes of severe LRTIs and 1732 of very severe LRTIs, of which 271 and 195, respectively, had RSV. Fifteen of 16 (94%) children with RSV died of LRTIs, 14 in the community and 1 in the hospital. The case fatality ratios for severe RSV LRTIs in the first 6 months of life were 3/52 (7.1%) and 1/36 (2.8%) in the community and hospital, respectively. Of those with very severe LRTIs in the community, 17.6% died. There were no very severe RSV LRTI hospital deaths. The adjusted RSV LRTI mortality rates ranged from 1.0 to 3.0/1000 child-years (CY) overall, and 2.0 to 6.1/1000 CY, accounting for 20% of the LRTI deaths and 10% of the postneonatal infant mortality. CONCLUSIONS: Community deaths from RSV account for the majority of RSV LRTI deaths, and efforts at prevention should be preferentially directed at populations where access to care is limited.

Efficacy of live attenuated and inactivated influenza vaccines among children in rural India: A 2-year, randomized, triple-blind, placebo-controlled trial.

BACKGROUND: Influenza is a cause of febrile acute respiratory infection (FARI) in India; however, few influenza vaccine trials have been conducted in India. We assessed absolute and relative efficacy of live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) among children aged 2 to 10 years in rural India through a randomized, triple-blind, placebo-controlled trial conducted over 2 years. METHODS AND FINDINGS: In June 2015, children were randomly allocated to LAIV, IIV, intranasal placebo, or inactivated polio vaccine (IPV) in a 2:2:1:1 ratio. In June 2016, vaccination was repeated per original allocation. Overall, 3,041 children received LAIV (n = 1,015), IIV (n = 1,010), nasal placebo (n = 507), or IPV (n = 509). Mean age of children was 6.5 years with 20% aged 9 to 10 years. Through weekly home visits, nasal and throat swabs were collected from children with FARI and tested for influenza virus by polymerase chain reaction. The primary outcome was laboratory-confirmed influenza-associated FARI; vaccine efficacy (VE) was calculated using modified intention-to-treat (mITT) analysis by Cox proportional hazards model (PH) for each year. In Year 1, VE was 40.0% (95% confidence interval (CI) 25.2 to 51.9) for LAIV and 59.0% (95% CI 47.8 to 67.9) for IIV compared with controls; relative efficacy of LAIV compared with IIV was -46.2% (95% CI -88.9 to -13.1). In Year 2, VE was 51.9% (95% CI 42.0 to 60.1) for LAIV and 49.9% (95% CI 39.2 to 58.7) for IIV; relative efficacy of LAIV compared with IIV was 4.2% (95% CI -19.9 to 23.5). No serious adverse vaccine-attributable events were reported. Study limitations include differing dosage requirements for children between nasal and injectable vaccines (single dose of LAIV versus 2 doses of IIV) in Year 1 and the fact that immunogenicity studies were not conducted. CONCLUSIONS: In this study, we found that LAIV and IIV vaccines were safe and moderately efficacious against influenza virus infection among Indian children. TRIAL REGISTRATION: Clinical Trials Registry of India CTRI/2015/06/005902.

Global burden of influenza-associated lower respiratory tract infections and hospitalizations among adults: A systematic review and meta-analysis.

BACKGROUND: Influenza illness burden is substantial, particularly among young children, older adults, and those with underlying conditions. Initiatives are underway to develop better global estimates for influenza-associated hospitalizations and deaths. Knowledge gaps remain regarding the role of influenza viruses in severe respiratory disease and hospitalizations among adults, particularly in lower-income settings. METHODS AND FINDINGS: We aggregated published data from a systematic review and unpublished data from surveillance platforms to generate global meta-analytic estimates for the proportion of acute respiratory hospitalizations associated with influenza viruses among adults. We searched 9 online databases (Medline, Embase, CINAHL, Cochrane Library, Scopus, Global Health, LILACS, WHOLIS, and CNKI; 1 January 1996-31 December 2016) to identify observational studies of influenza-associated hospitalizations in adults, and assessed eligible papers for bias using a simplified Newcastle-Ottawa scale for observational data. We applied meta-analytic proportions to global estimates of lower respiratory infections (LRIs) and hospitalizations from the Global Burden of Disease study in adults ≥20 years and by age groups (20-64 years and ≥65 years) to obtain the number of influenza-associated LRI episodes and hospitalizations for 2016. Data from 63 sources showed that influenza was associated with 14.1% (95% CI 12.1%-16.5%) of acute respiratory hospitalizations among all adults, with no significant differences by age group. The 63 data sources represent published observational studies (n = 28) and unpublished surveillance data (n = 35), from all World Health Organization regions (Africa, n = 8; Americas, n = 11; Eastern Mediterranean, n = 7; Europe, n = 8; Southeast Asia, n = 11; Western Pacific, n = 18). Data quality for published data sources was predominantly moderate or high (75%, n = 56/75). We estimate 32,126,000 (95% CI 20,484,000-46,129,000) influenza-associated LRI episodes and 5,678,000 (95% CI 3,205,000-9,432,000) LRI hospitalizations occur each year among adults. While adults <65 years contribute most influenza-associated LRI hospitalizations and episodes (3,464,000 [95% CI 1,885,000-5,978,000] LRI hospitalizations and 31,087,000 [95% CI 19,987,000-44,444,000] LRI episodes), hospitalization rates were highest in those ≥65 years (437/100,000 person-years [95% CI 265-612/100,000 person-years]). For this analysis, published articles were limited in their inclusion of stratified testing data by year and age group. Lack of information regarding influenza vaccination of the study population was also a limitation across both types of data sources. CONCLUSIONS: In this meta-analysis, we estimated that influenza viruses are associated with over 5 million hospitalizations worldwide per year. Inclusion of both published and unpublished findings allowed for increased power to generate stratified estimates, and improved representation from lower-income countries. Together, the available data demonstrate the importance of influenza viruses as a cause of severe disease and hospitalizations in younger and older adults worldwide.

Global circulation patterns of seasonal influenza viruses vary with antigenic drift.

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

Laboratory-Confirmed Avian Influenza A(H9N2) Virus Infection, India, 2019.

A 17-month-old boy in India with severe acute respiratory infection was laboratory confirmed to have avian influenza A(H9N2) virus infection. Complete genome analysis of the strain indicated a mixed lineage of G1 and H7N3. The strain also was found to be susceptible to adamantanes and neuraminidase inhibitors.

Molecular characterization of influenza A(H1N1)pdm09 viruses circulating at various geographical locations in India, 2017.

Background & objectives: Influenza virological surveillance is an essential tool for the early detection of novel genetic variants of epidemiologic and clinical significance. This study was aimed to genetically characterize A(H1N1)pdm09 virus circulating in 2017 and to compare it with the global data. Methods: The regional/State Viral Research and Diagnostic Laboratories (VRDLs) provided influenza diagnosis for referred clinical samples and shared influenza A(H1N1)pdm09 positives with the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India, for hemagglutinin (HA) gene phylogenetic analysis. Sites at Manipal, Jaipur and Dibrugarh performed the sequencing and shared the sequence data for analysis. The antiviral susceptibility of influenza viruses was assessed for known molecular marker H275Y at the ICMR-NIV, Pune. Results: All the eight VRDLs had well-established influenza diagnostic facilities and showed increased activity of influenza A(H1N1)pdm09 during 2017. Phylogenetic analysis showed that the viruses from the different regions of the country were similar to A/Michigan/45/2015 strain which was the 2017-2018 recommended vaccine strain and were clustered with the globally circulating clade 6B.1 with signature mutations S84N, S162N and I216T. The clade 6B.1 showed further subgrouping with additional mutations S74R, S164T and I295V; however, there was no significant association between the presence of these mutations and severity of disease due to influenza. All the study viruses were sensitive to oseltamivir. Interpretation & conclusions: During the study period, all the study sites reported globally circulating A/Michigan/45/2015 vaccine strain of influenza A(H1N1)pdm09 viruses and remained sensitive to oseltamivir. Further genetic and antigenic characterization of influenza viruses is recommended to address public health concerns.

Emerging/re-emerging viral diseases & new viruses on the Indian horizon.

Infectious diseases remain as the major causes of human and animal morbidity and mortality leading to significant healthcare expenditure in India. The country has experienced the outbreaks and epidemics of many infectious diseases. However, enormous successes have been obtained against the control of major epidemic diseases, such as malaria, plague, leprosy and cholera, in the past. The country's vast terrains of extreme geo-climatic differences and uneven population distribution present unique patterns of distribution of viral diseases. Dynamic interplays of biological, socio-cultural and ecological factors, together with novel aspects of human-animal interphase, pose additional challenges with respect to the emergence of infectious diseases. The important challenges faced in the control and prevention of emerging and re-emerging infectious diseases range from understanding the impact of factors that are necessary for the emergence, to development of strengthened surveillance systems that can mitigate human suffering and death. In this article, the major emerging and re-emerging viral infections of public health importance have been reviewed that have already been included in the Integrated Disease Surveillance Programme.

Seroprevalence of influenza A H1N1 (swine) infection in the human population in a cantonment.

BACKGROUND: Various serosurveys and studies were conducted globally on pandemic influenza. H1N1 virus reported so far provides ample evidence of differing perspectives, regarding its epidemiology especially with regard to prevalence, populations groups, and behaviour related to vaccine acceptance. A multigroup, cross-sectional survey among 658 healthy subjects was carried out, in Pune among students, health-care workers (HCWs), and soldiers to assess the seroprevalence of pandemic influenza H1N1 virus and its associated factors. METHODS: The total sample size, based on forecasted prevalence of 33%, worked out to be 640. We studied 658 subjects including 103 students, 201 HCWs, and 354 serving soldiers. The sample for each group was selected from the respective study population by simple random sampling using a random number table. Haemagglutination inhibition test was carried out at the National Institute of Virology. RESULTS: The overall seroprevalence of pandemic influenza H1N1 (2009) virus was found to be 46.5% (95% confidence interval 42.6-50.4) which was adjusted to 39.4% after excluding those vaccinated. The availability of vaccine for high-risk group such as HCWs did not find much favour with the HCWs who did not accept vaccine for various reasons. Whereas only one student was vaccinated, 21.4% of HCWs and 32.5% of soldiers were vaccinated. CONCLUSION: Based on high seroprevalence of antibodies against H1N1 virus during pandemic, vaccination of general population is not recommended. However, high-risk groups and HCWs need to be protected with flu vaccine. There is a need to encourage HCWs for accepting vaccination.

Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study.

BACKGROUND: We have previously estimated that respiratory syncytial virus (RSV) was associated with 22% of all episodes of (severe) acute lower respiratory infection (ALRI) resulting in 55 000 to 199 000 deaths in children younger than 5 years in 2005. In the past 5 years, major research activity on RSV has yielded substantial new data from developing countries. With a considerably expanded dataset from a large international collaboration, we aimed to estimate the global incidence, hospital admission rate, and mortality from RSV-ALRI episodes in young children in 2015. METHODS: We estimated the incidence and hospital admission rate of RSV-associated ALRI (RSV-ALRI) in children younger than 5 years stratified by age and World Bank income regions from a systematic review of studies published between Jan 1, 1995, and Dec 31, 2016, and unpublished data from 76 high quality population-based studies. We estimated the RSV-ALRI incidence for 132 developing countries using a risk factor-based model and 2015 population estimates. We estimated the in-hospital RSV-ALRI mortality by combining in-hospital case fatality ratios with hospital admission estimates from hospital-based (published and unpublished) studies. We also estimated overall RSV-ALRI mortality by identifying studies reporting monthly data for ALRI mortality in the community and RSV activity. FINDINGS: We estimated that globally in 2015, 33·1 million (uncertainty range [UR] 21·6-50·3) episodes of RSV-ALRI, resulted in about 3·2 million (2·7-3·8) hospital admissions, and 59 600 (48 000-74 500) in-hospital deaths in children younger than 5 years. In children younger than 6 months, 1·4 million (UR 1·2-1·7) hospital admissions, and 27 300 (UR 20 700-36 200) in-hospital deaths were due to RSV-ALRI. We also estimated that the overall RSV-ALRI mortality could be as high as 118 200 (UR 94 600-149 400). Incidence and mortality varied substantially from year to year in any given population. INTERPRETATION: Globally, RSV is a common cause of childhood ALRI and a major cause of hospital admissions in young children, resulting in a substantial burden on health-care services. About 45% of hospital admissions and in-hospital deaths due to RSV-ALRI occur in children younger than 6 months. An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group. FUNDING: The Bill & Melinda Gates Foundation.

Respiratory viruses in returning Hajj & Umrah pilgrims with acute respiratory illness in 2014-2015.

BACKGROUND & OBJECTIVES: Respiratory tract infections are common among Hajj and Umrah pilgrims which pose a public health risk of spread of respiratory infections. Influenza has been reported from Indian Hajj and Umrah returning pilgrims, but data on other respiratory pathogens are sparse in India. Here we report the presence of common respiratory viral pathogens in returning Hajj and Umrah pilgrims suffering from acute respiratory illness (ARI) in 2014-2015. METHODS: Respiratory specimens (nasopharyngeal and throat swabs) were collected from 300 consenting pilgrims with ARI in the past one week and tested for influenza and Middle East Respiratory Syndrome coronavirus (MERS-CoV) and other respiratory viruses using in-house standardized quantitative real-time reverse-transcription polymerase chain reaction. Clinical features among the pathogen positive and negative patients were compared. The patients received symptomatic treatment and antivirals where appropriate and were followed telephonically to collect data on illness outcome. RESULTS: Ninety seven (32.3%) of the 300 participants were tested positive for any virus, most common being influenza viruses (n=33, 11%). Other respiratory viruses that were detected included human coronaviruses [n=26, 8.7%; OC43 (n=19, 6.3%) and C229E (n=7, 2.3%)], rhinovirus (n=20, 6%), adenoviruses (n=8, 2.6%), parainfluenza viruses (n=7, 2.3%), respiratory syncytial virus (n=3, 1%) and bocaviruses (n=2, 0.6%). Clinical features observed in pathogen positive and pathogen negative patients did not differ significantly. Eighteen influenza positive patients were treated with oseltamivir. INTERPRETATION & CONCLUSIONS: Pilgrims returning from mass gatherings are often afflicted with respiratory pathogens with a potential to facilitate transmission of respiratory pathogens across international borders. The study reinforces the need for better infection prevention and control measures such as vaccination, health education on cough etiquette and hand hygiene.

Retrospective analysis of clinical information in Crimean-Congo haemorrhagic fever patients: 2014-2015, India.

BACKGROUND & OBJECTIVES: Differential diagnosis of Crimean-Congo haemorrhagic fever (CCHF) from other acute febrile illnesses with haemorrhagic manifestation is challenging in India. Nosocomial infection is a significant mode of transmission due to exposure of healthcare workers to blood and body fluids of infected patients. Being a risk group 4 virus, laboratory confirmation of infection is not widely available. In such a situation, early identification of potential CCHF patients would be useful in limiting the spread of the disease. The objective of this study was to retrospectively analyse clinical and laboratory findings of CCHF patients that might be useful in early detection of a CCHF case in limited resource settings. METHODS: Retrospective analysis of clinical and laboratory data of patients suspected to have CCHF referred for diagnosis from Gujarat and Rajasthan States of India (2014-2015) was done. Samples were tested using CCHF-specific real time reverse transcription (RT)-PCR and IgM ELISA. RESULTS: Among the 69 patients referred, 21 were laboratory confirmed CCHF cases of whom nine had a history of occupational exposure. No clustering of cases was noted. Platelet count cut-off for detection of positive cases by receiver operating characteristic curve was 21.5×10[9]/l with sensitivity 82.4 per cent and specificity 82.1 per cent. Melaena was a significant clinical presentation in confirmed positive CCHF patients. INTERPRETATION & CONCLUSIONS: The study findings suggest that in endemic areas thrombocytopenia and melaena may be early indicators of CCHF. Further studies are needed to confirm these findings.

Global Role and Burden of Influenza in Pediatric Respiratory Hospitalizations, 1982-2012: A Systematic Analysis.

BACKGROUND: The global burden of pediatric severe respiratory illness is substantial, and influenza viruses contribute to this burden. Systematic surveillance and testing for influenza among hospitalized children has expanded globally over the past decade. However, only a fraction of the data has been used to estimate influenza burden. In this analysis, we use surveillance data to provide an estimate of influenza-associated hospitalizations among children worldwide. METHODS AND FINDINGS: We aggregated data from a systematic review (n = 108) and surveillance platforms (n = 37) to calculate a pooled estimate of the proportion of samples collected from children hospitalized with respiratory illnesses and positive for influenza by age group (<6 mo, <1 y, <2 y, <5 y, 5-17 y, and <18 y). We applied this proportion to global estimates of acute lower respiratory infection hospitalizations among children aged <1 y and <5 y, to obtain the number and per capita rate of influenza-associated hospitalizations by geographic region and socio-economic status. Influenza was associated with 10% (95% CI 8%-11%) of respiratory hospitalizations in children <18 y worldwide, ranging from 5% (95% CI 3%-7%) among children <6 mo to 16% (95% CI 14%-20%) among children 5-17 y. On average, we estimated that influenza results in approximately 374,000 (95% CI 264,000 to 539,000) hospitalizations in children <1 y-of which 228,000 (95% CI 150,000 to 344,000) occur in children <6 mo-and 870,000 (95% CI 610,000 to 1,237,000) hospitalizations in children <5 y annually. Influenza-associated hospitalization rates were more than three times higher in developing countries than in industrialized countries (150/100,000 children/year versus 48/100,000). However, differences in hospitalization practices between settings are an important limitation in interpreting these findings. CONCLUSIONS: Influenza is an important contributor to respiratory hospitalizations among young children worldwide. Increasing influenza vaccination coverage among young children and pregnant women could reduce this burden and protect infants <6 mo.

Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

Differences in influenza seasonality by latitude, northern India.

The seasonality of influenza in the tropics complicates vaccination timing. We investigated influenza seasonality in northern India and found influenza positivity peaked in Srinagar (34.09°N) in January-March but peaked in New Delhi (28.66°N) in July-September. Srinagar should consider influenza vaccination in October-November, but New Delhi should vaccinate in May-June.

Molecular characterization of circulating pandemic strains of influenza A virus during 2012 to 2013 in Lucknow (India).

The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populations. However, it is still causing sporadic outbreaks worldwide with different severity. The present study was planned to understand the genetic diversity (based on the HA1 gene) of influenza A(H1N1)pdm09 strains circulating during the post pandemic period. The HA1 gene was selected because the HA1 protein is immunogenic, functions as a receptor binding site and indirectly affects transmission and pathogenicity of virus. A total of 2,818 cases were enrolled. Nasal/throat swabs from all cases were tested by one-step real time PCR for detection of influenza virus types and subtypes according to the CDC protocol. Of these, 134 cases were A(H1N1)pdm09 positive, 34 of which were screened for HA1 gene (position 434-905) sequencing (Big-Dye terminator using 3130 ABI, Genetic analyzer). Molecular and phylogenetic analysis was performed using PhyML approach (v. 3.0). All A(H1N1)pdm09 positive and negative cases were clinically characterized. Phylogentically, all Lucknow strains (n = 33) except one fall with the clade seven reference strain. One strain showed 99.9% similarities with clade one reference strain A/California/07/2009. In mutational analysis, 33 strains had the S220T mutation, which is at an antigenic site and characteristic of clade seven along with few minor mutations; K180I/T/Q, V190I, S200P, S202T, A203T, A214T, S220T, V251I, and A273T. These results suggest that clade seven was the most widely circulating clade in Lucknow and A(H1N1)pdm09 cases showed mild clinical symptoms as compared to A(H3N2) or influenza B cases.

Influenza seasonality and vaccination timing in tropical and subtropical areas of southern and south-eastern Asia.

OBJECTIVE: To characterize influenza seasonality and identify the best time of the year for vaccination against influenza in tropical and subtropical countries of southern and south-eastern Asia that lie north of the equator. METHODS: Weekly influenza surveillance data for 2006 to 2011 were obtained from Bangladesh, Cambodia, India, Indonesia, the Lao People's Democratic Republic, Malaysia, the Philippines, Singapore, Thailand and Viet Nam. Weekly rates of influenza activity were based on the percentage of all nasopharyngeal samples collected during the year that tested positive for influenza virus or viral nucleic acid on any given week. Monthly positivity rates were then calculated to define annual peaks of influenza activity in each country and across countries. FINDINGS: Influenza activity peaked between June/July and October in seven countries, three of which showed a second peak in December to February. Countries closer to the equator had year-round circulation without discrete peaks. Viral types and subtypes varied from year to year but not across countries in a given year. The cumulative proportion of specimens that tested positive from June to November was > 60% in Bangladesh, Cambodia, India, the Lao People's Democratic Republic, the Philippines, Thailand and Viet Nam. Thus, these tropical and subtropical countries exhibited earlier influenza activity peaks than temperate climate countries north of the equator. CONCLUSION: Most southern and south-eastern Asian countries lying north of the equator should consider vaccinating against influenza from April to June; countries near the equator without a distinct peak in influenza activity can base vaccination timing on local factors.

Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.

Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.