[Show abstract][Hide abstract] ABSTRACT: Abstract:
Background and Objective:
Recent antiviral studies from South East Asia, Europe and the United States showed the presence of neuraminidase inhibitor (NAIs) resistance in pandemic influenza 2009 viruses. The study was undertaken to evaluate neuraminidase inhibitor (NAI) resistance of pandemic Influenza virus isolated from India.
Pandemic Influenza viruses, isolated from 2009 to 2013 were analyzed genetically for known resistance markers in NA gene. Pandemic (H1N1) (n= 493) isolates were tested for H274Y mutation by rRT-PCR. Randomly selected resistant and sensitive viruses were confirmed by phenotypic assay.
All Pandemic A/H1N1 isolates remained sensitive except single 2013 isolate. Genetic analysis of single 2013 isolate as well as original clinical material showed H274Y mutation responsible for reduce susceptibility to Oseltamivir and were also confirmed by phenotypic assay.
Emergence of pandemic influenza strain resistant to oseltamivir emphasizes the need for monitoring antiviral resistance as part of National Influenza Program
WHO South East Journal Of Public health. 01/2014; In Press.
[Show abstract][Hide abstract] ABSTRACT: Human respiratory syncytial virus (RSV) is one of the most important respiratory viruses causing acute respiratory tract infections amongst children. Based on genotyping of the attachment glycoprotein (G) gene, it is divided into two groups, RSV-A and RSV-B. Infection with one group does not confer immunity against the other and children infected with one antigenic group are more likely to be reinfected with the heterologous group. We tested 854 samples of patients with influenza like illness (ILI)/ severe respiratory illness (SARI) during the period 2009-2012 for RSV using a conventional multiplex RT-PCR and found 159 (18.61%) samples to be positive for RSV of which 130 (15.22%) were positive for RSV-B and 29 (3.39%) for RSV-A suggesting that RSV-B was the predominant group circulating in western India during the study period. Seasonal RSV outbreaks were observed in the monsoon and winter months. RSV was more prevalent amongst children in the 0-24 month age group (21.53%) in comparison to children in the 24-60 month age group (13.01%). Phylogenetic analysis using the G gene of 27 representative RSV-A positive samples revealed that all sequences belonged to the NA1 genotype. Of these, 5 sequences exhibited the novel 72 nucleotide duplication in the C-terminal of the G gene first reported from Ontario, Canada and clustered in the newly designated ON1 genotype. Also, 32 of the 33 RSV-B sequences exhibited the 60 nucleotide duplication associated with genotype BA and phylogenetic analysis showed that these sequences belonged to the genotype BA9 and BA12. We also found one RSV-B sequence belonging to genotype GB2, which has not been previously reported in India.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10/2013; · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Archives of Virology 08/2013; · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. Multiplex Reverse Transcriptase Polymerase Chain Reaction (mRT-PCR) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. Objective of the present study was to develop a one-step mRT-PCR that could detect 18 respiratory viruses in three sets. The method was compared with real time RT-PCR (rRT-PCR) for its sensitivity and specificity. Clinical specimens from 843 pediatric patients with respiratory symptoms were used in the study. 503 (59.7%) samples were detected positive by mRT-PCR. Of these 462 (54.8%) exhibited presence of a single pathogen and 41 (4.9%) had multiple pathogens. rRT-PCR detected 439 (52.1%) positive samples, where 419 (49.7%) exhibited one virus and 20 (2.4%) showed co-infections. Concordance between mRT-PCR and rRT-PCR was 91.9% and kappa correlation 0.837. Sensitivity and specificity of mRT-PCR was 99.5% and 83.7% while that of rRT-PCR was 86.9% and 99.4% respectively. Rhinovirus (17.2%) was the most frequently detected virus followed by respiratory syncytial virus B (15.4%), H1N1pdm09 (8.54%), parainfluenza virus -3 (5.8%) and metapneumovirus (5.2%). In conclusion, mRT-PCR is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses.
Journal of virological methods 01/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Systematic influenza virus surveillance has been carried out in India since 2004 and has revealed the cocirculation of type B lineages. The genetic diversity of influenza B viruses was observed when full-genome analysis was performed. In 2010, the cocirculation of multiple genotypes was observed.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND and OBJECTIVE:
Recent influenza antiviral resistance studies in South East Asia, Europe and the United States reveal adamantane and neuraminidase inhibitor (NAIs) resistance. The study was undertaken to evaluate antiviral resistance of Influenza viruses isolated from various parts of India.
METHODS: Influenza viruses, isolated from 2004 to 2011 were analyzed genetically for known resistance markers byM2 and NA gene sequencing. Influenza A/H1N1 (n=206), A/H3N2 (n=371) viruses for amantadine resistance and A/H1N1 (n=206), A/H3N2 (n=272) and Type B (n=326) for oseltamivir resistance were sequenced. Pandemic (H1N1) (n= 493) isolates were tested for H274Y mutation by rRT-PCR. Randomly selected resistant and sensitive influenza A/ H1N1 and A/H3N2 viruses were confirmed by phenotypic assay.
Serine to asparagine (S3IN) mutation was detected in six isolates of 2007-2008.One dual-resistant A/H1N1 was detected for the first time in India with leucine to phenylalanine (L26F) mutation in M2 gene and H274Y mutation in NA gene. A/H3N2 viruses showed an incremental trend in resistance to amantadine from 22.5% in 2005 to 100% in 2008 onwards with S3IN mutation. Fifty of the sixty one (82%) A/H1N1 viruses tested in 2008-2009 were oseltamivir resistant with H274Y mutation, while all A/H3N2, pandemic A/H1N1 and Type B isolates remained sensitive. Genetic results were also confirmed by phenotypic analysis of randomly selected 50 resistant A/ H1N1 and 40 sensitive A/H3N2 isolates.
Emergence of influenza strains resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for monitoring antiviral resistance as part of National Influenza Program
The Indian Journal of Medical Research 01/2013; In Press. · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza is vaccine-preventable; however, the burden of severe influenza in India remains unknown. We conducted a population-based study to estimate the incidence of laboratory confirmed influenza-associated hospitalizations in a rural community in western India.
We conducted active surveillance for hospitalized patients with acute medical illnesses or acute chronic disease exacerbations in Pune during pandemic and post pandemic periods (May 2009-April 2011). Nasal and throat swabs were tested for influenza viruses. A community health utilization survey estimated the proportion of residents hospitalized with respiratory illness at non-study facilities and was used to adjust incidence estimates from facility-based surveillance.
Among 9,426 hospitalizations, 3,391 (36%) patients were enrolled; 665 of 3,179 (20.9%) tested positive for influenza. Of 665 influenza positives, 340 (51%) were pandemic A(H1N1)pdm09 and 327 (49%) were seasonal, including A/H3 (16%), A/H1 (3%) and influenza B (30%). The proportion of patients with influenza peaked during August 2009 (39%) and 2010 (42%). The adjusted annual incidence of influenza hospitalizations was 46.8/10,000 during pandemic and 40.5/10,000 during post-pandemic period with comparable incidence of A(H1N1)pdm09 during both periods (18.8 and 20.3, respectively). The incidence of both pH1N1 and seasonal hospitalized influenza disease was highest in the 5-29 year olds.
We document the previously unrecognized burden of influenza hospitalization in a rural community following the emergence of influenza A(H1N1)pdm09 viruses in India. During peak periods of influenza activity circulation i.e during the monsoon period, 20% of all hospital admissions in the community had influenza positivity. These findings can inform development of influenza prevention and control strategies in India.
PLoS ONE 01/2013; 8(5):e55918. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two complete genomes of human respiratory syncytial virus subtype A (HRSV-A), with and without a 72-nucleotide duplication in the C-terminal glycoprotein G gene, were sequenced and analyzed. Characterization of these genomes will improve understanding of the diversity, emergence, virulence, pathogenicity, and transmissibility of a novel RSV-A genotype with a 72-nucleotide G gene duplication.
[Show abstract][Hide abstract] ABSTRACT: Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important.
55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide.
Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains.
Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.
[Show abstract][Hide abstract] ABSTRACT: An explosive outbreak of Hepatitis B with high mortality was reported in 2009, in Modasa, Gujarat, India. Mortality was associated with basal core promoter and precore mutant hepatitis B virus (HBV). The current study addresses the role of immunological parameters in the progression to fulminant hepatitis. The study population comprised of 22 acute HBV patients, 13 fulminant HBV liver failure patients and 54 healthy controls. Hepatitis B surface antigen-induced CTL responses by enzyme-linked immunosorbent spot (ELISPOT), cytokine and chemokine quantitation by Bioplex assay, peripheral NK, natural killer T (NKT), CD4 and CD8 T-cell frequencies by flow cytometry were carried out. The median percentage of NK cells in the lymphocytes of the acute and fulminant liver failure patients were significantly lower compared to controls. Acute and fulminant liver failure patients had significantly high and comparable NKT cells compared to controls, respectively. Importantly, NKT cells were significantly lower in fulminant HBV liver failure than acute HBV patients. Circulating peripheral CD4/CD8 T-cell subsets among the patient categories and controls were comparable. In acute HBV patients, a significant increase in IFN-γ release was recorded (ELISPOT) by the unstimulated, antigen-stimulated and mitogen-stimulated cells when compared to controls. Comparisons of cytokines and chemokines among the disease categories revealed significantly lower levels of CCL4 in fulminant liver failure patients. NKT cells and CCL4 might be playing a pivotal role in limiting HBV infection among the patients investigated.
Journal of Viral Hepatitis 10/2011; 18(10):e415-22. · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza surveillance is important to identify circulating, emerging/reemerging strains and unusual epidemiological trends. With these objectives, a multisite human influenza surveillance network was initiated in India in 2004.
Epidemiologic data and throat swabs for laboratory testing were collected from patients with influenza-like illness (ILI) and severe acute respiratory infections (SARI). Virus isolation was carried out in Madin-Darby canine kidney cells and strains identified by hemagglutination inhibition assay. Meteorological data were collected.
From September 2004 to December 2008, 617 (4·43%) of 13928 cases yielded isolates: 27·8% were influenza A(H1N1), 29·8% were type A(H3N2), and 42·3% were type B. The yearly type and subtype distribution varied significantly from site to site. Peak influenza activity was observed from June to August in Delhi, Pune, and Kolkata and October to December in Chennai. Maximum influenza activity was seen during the rains in Delhi, Pune, Chennai, and Kolkata in correlation with virus isolations. Multivariate analysis of ILI cases showed chill/rigors, cough, fatigue, and ILI in family, correlated positively with isolation. Genetic analysis of Indian isolates revealed that viruses matched with vaccine strains by and large. Overlapping between circulating and vaccine component strains of consecutive years was also observed.
Seasonal influenza A(H1N1), H3N2, and type B co-circulated in all regions without any particular pattern of movement of any subtype. Year-round limited influenza activity with peaks during rains was observed. Genetic drifts and varying seasonality in different parts of the country suggest that a staggered timing of vaccination may be appropriate for India.
Influenza and Other Respiratory Viruses 09/2011; 6(3):196-203. · 1.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute respiratory illness (ARI) is one of the major health problems in tropical countries of Asia, like India where approximately 0.5 million children in the age group of < 5 years die annually. Previously we have reported the genetic characterization of influenza A (Inf-A) strains circulating in Kolkata, eastern India. This study was initiated to characterize the genetic diversity of the circulating influenza B (Inf-B) viruses. Of 3035 nasal/throat swabs, 494 (16.3%) samples were identified as influenza A/B positive by real time RT-PCR, of which 244 samples were confirmed having Inf-B infection. Comparison of nucleotide (nt) and amino acid (aa) sequences of HA and NA gene of Inf-B viruses revealed co-circulation of B/Yamagata and B/Victoria lineages. Of the 32 randomly selected Inf-B strains from Kolkata, seventeen strains possessed reassorted NA gene. There was a single Histidine to Asparagine substitution in the 131st position which is a part of 120 loop on HA1 region along with a deletion at position 178 in the Kolkata strains belonging to the Yamagata lineage. Amino acid substitution was observed at position 198 on NA gene in the strains B/Kol/542/2006, B/Kol/1373/2008, B/Kol/1880/2008, B/Kol/2044/2008 and in all the representative strains isolated during 2009 with respect to the circulating vaccine strains. This substitution is responsible for reduced sensitivity of neuraminidase inhibitors. The results highlight the importance of monitoring Inf-B viruses for development of antiviral resistance among circulating strains.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 06/2011; 11(7):1595-601. · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The indigenous transmission of the 2009 pandemic H1N1 (pH1N1) virus in India made it as one of the major sub-types in circulation. Genetic characterization indicated that the viruses predominantly clustered in clade 7, the globally most widely circulating pH1N1 clade. It is imperative to continue monitoring the genetic make-up of the pH1N1 viruses to understand their adaptability and evolutionary dynamics in the country. We characterized 31 full genomes and 94 hemagglutinin (HA) sequences of the pH1N1 viruses from various regions of India (May 2009-October 2010). Among the newly identified mutations reported in the pH1N1 viruses that could alter the viral fitness, E374K in the HA was increasingly noted in 35 Indian isolates beyond September 2009 and its co-occurrence with D97N or V30A was also observed in the more recent isolates. Molecular clock analysis based on all Indian isolates and closely related global representatives indicated higher substitution rates (∼ 7.1 × 10(-3) subs/site/year) when compared to an earlier report. Several independent introductions were noted within the country along with considerable evidence of indigenous evolution during the latter period of the study. The estimate for the mean age of the common ancestor of all the pandemic isolates dated to around August 2008 correlating well with the global estimate. Evidence for adaptive evolution in the HA was observed in the clade 7 isolates at the 'Ca' antigenic site that may have implications for future re-evaluation of the vaccine composition. The study thus warrants the need for continued surveillance and genetic characterization of whole genome sequences to detect any possible reassortment events that might further contribute to the viral fitness of the pH1N1 viruses.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 03/2011; 11(5):997-1005. · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral shedding profile of infections caused by the novel influenza A (pH1N1) virus has not been
extensively studied. In the present study we aimed to compare the influenza viral load in clinical
specimens collected from different body sites of patients to analyze the best specimen for detecting
viral load and predicting disease severity. The respiratory specimens (throat and nasal swabs), urine
and serum were collected from patients on first day of their hospital visit within 48 h of onset of
influenza like illness (ILI) and screened for influenza positivity in respiratory specimens by real-time RTPCR.
A total of 10 pandemic H1N1 and 15 seasonal influenza positive cases were included in this study
and viral load was estimated in all the types of specimens by real-time RT-PCR. Our findings revealed
that the nasal swab had the highest mean viral load of 21.406 x 104 followed by throat swab (12.777 x
104), urine (0.026 x 104), serum (0.0007 x 104). These findings confirm that nasal secretions are the best
specimen, followed by throat swab, urine and serum. The importance of this study is to show the viral
shedding profile in different specimen types and to suggest alternatives to respiratory specimens for
the diagnosis of influenza.
International Journal of Medicine and Medical Sciences. 01/2011; 3:144-148.
[Show abstract][Hide abstract] ABSTRACT: Influenza surveillance was implemented in Kolkata, eastern India in 2005 to identify the circulating subtypes and characterize their genetic diversity. Throat and nasal swabs were collected from outpatients with influenza-like illness (ILI). Of 2844 ILI cases identified at two referral hospitals during October 2005-September 2009, 309 (10.86%) were positive for Influenza A by real time RT-PCR, of which 110 (35.60%) were subtyped as H1N1 and 199 (64.40%) as H3N2. Comparison of the nucleotide (nt) and amino acid (aa) sequences of the HA1 gene for H1N1 and H3N2 strains showed that a subset of strains precede WHO recommended contemporary strains by 1-2 years. The Kolkata H1N1 strains clustered in Clade II, subgroup 2B with A/Brisbane/59/2007 but were distant from the corresponding vaccine strains (New Caledonia/20/99 and A/Solomon Island/3/06). The 2005-06 and 2007 H3N2 strains (15/17) clustered either A/Brisbane/10/2007-like (n=8) or A/Nepal/921/2006 like (n=7) strains, whereas 2008 strains (8/12) and 2009 strains (4/4) were similar to the 2010-11 vaccine strain A/Perth/16/2009. More aa substitutions were found in HA or NA genes of H3N2 than in H1N1 strains. No mutation conferring neuraminidase resistance was observed in any of the strain during 2005-08, however in 2009, drug resistant marker (H275Y) was present in seasonal H1N1, but not in co-circulating H3N2 strains. This is the first report of genetic characterization of circulating Influenza A strains from India. The results also highlight the importance of continuing Influenza surveillance in developing countries of Asia for monitoring unusual strains with pandemic potential and mutations conferring antiviral resistance.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 12/2010; 10(8):1188-98. · 3.22 Impact Factor