Influenza A(H1N1)pdm09 outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in Pune, India, 2012-2015.

An outbreak of influenza A(H1N1)pdm09 was detected during the ongoing community-based surveillance of influenza-like illness (ILI). Among reported 119 influenza A(H1N1)pdm09 cases (59 cases in the year 2012 and 60 cases in 2015) in summer months, common clinical features were fever (100%), cough (90·7%), sore throat (85·7%), nasal discharge (48·7%), headache (55·5%), fatigue (18·5%), breathlessness (3·4%), and ear discharge (1·7%). Rise in ILI cases were negatively correlated with the seasonal factors such as relative humidity (Karl Pearson's correlation coefficient, i.e. r = -0·71 in the year 2012 and r = -0·44 in the year 2015), while rise in ILI cases were positively correlated with the temperature difference (r = 0·44 in the year 2012 and r = 0·77 in the year 2015). The effective reproduction number R, was estimated to be 1·30 in 2012 and 1·64 in 2015. The study highlights the rise in unusual influenza activity in summer month with high attack rate of ILI among children aged ⩽9 years. Children in this age group may need special attention for influenza vaccination. Influenza A(H1N1)pdm09 outbreak was confirmed in inter-seasonal months during the surveillance of ILI in Pune, India, 2012-2015.

Antiviral drug profile of human influenza A & B viruses circulating in India: 2004-2011.

BACKGROUND & OBJECTIVES: Recent influenza antiviral resistance studies in South East Asia, Europe and the United States reveal adamantane and neuraminidase inhibitor (NAIs) resistance. This study was undertaken to evaluate antiviral resistance in influenza viruses isolated from various parts of India, during 2004 to 2011. METHODS: Influenza viruses were analyzed genetically for known resistance markers by M2 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 real time reverse transcription (rRT)-PCR. Randomly selected resistant and sensitive influenza A/H1N1 and A/H3N2 viruses were confirmed by phenotypic assay. RESULTS: 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 increase in resistance to amantadine from 22.5 per cent in 2005 to 100 per cent in 2008 onwards with S3IN mutation. Fifty of the 61 (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. INTERPRETATION & CONCLUSIONS: Emergence of influenza viruses resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for continuous monitoring of antiviral resistance.

Epidemic of hepatitis B with high mortality in India: association of fulminant disease with lack of CCL4 and natural killer T cells.

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.

An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes.

In 2009, an outbreak of hepatitis B with high mortality was observed in Sabarkantha district, Gujarat state, India with 456 cases and 89 deaths. Hospitalized patients with self-limiting disease (152, AVH)) and fulminant hepatic failure (39, FHF including 27 fatal and 12 survivals) were investigated. These were screened for diagnostic markers for hepatitis viruses, hepatitis B virus (HBV) genotyping and mutant analysis. Complete HBV genomes from 22 FHF and 17 AVH cases were sequenced. Serosurveys were carried out in the most and least affected blocks for the prevalence of HBV and identification of mutants. History of injection from a physician was associated with FHF and AVH cases. Co-infection with other hepatitis viruses or higher HBV DNA load was not responsible for mortality. Four blocks contributed to 85.7% (391/456) of the cases and 95.5% (85/89) mortality while two adjacent blocks had negligible mortality. Sequence analysis showed the presence of pre-core and basal core promoter mutants and 4 amino acid substitutions exclusively among FHF cases. None of the self-limiting patients exhibited these dual mutations. Genotype D was predominant, D1 being present in all FHF cases while D2 was most prevalent in AVH cases. Probably due to violation of accepted infection control procedures by the qualified medical practitioners, HBV prevalence was higher in the affected blocks before the outbreak. Gross and continued use of HBV contaminated (mutant and wild viruses) injection devices led to an explosive outbreak with high mortality with a striking association with pre-C/BCP mutants and D1 genotype.

Diosgenin and beta-Sitosterol: Isolation from Solanum Xanthocarpum Tissue Cultures.

Diosgenin and beta-sitosterol were isolated from Solanum xanthocarpum callus, crystallized, and chemically characterized. That these metabolites, particularly diosgenin, form in significant amounts in tissue culture may prove useful.

Viral load, antibody titers and recombinant open reading frame 2 protein-induced TH1/TH2 cytokines and cellular immune responses in self-limiting and fulminant hepatitis e.

OBJECTIVES: Hepatitis E virus (HEV) is the predominant cause of water-borne epidemics, sporadic acute viral hepatitis (AVH) in adults and fulminant hepatic failure (FHF) among pregnant women and other adults in India. This preliminary study was designed to examine the association of viral load and certain host immune responses with uneventful recovery or progression to FHF. METHODS: Viral load, anti-HEV antibody titers, rORF2p-induced Th1/Th2 cytokines levels and cellular immune responses were assessed in 47 patients with self-limiting hepatitis E and 14 FHF-E cases. The controls included 16 anti-HEV-IgM and IgG-negative healthy individuals. RESULTS: In AVH category, the viral load was 2.4 x 10(4) +/- 1.92 x 10(4) copies/ml while except for one, all FHF patients were negative for HEV RNA; anti-HEV-IgM and IgG titers were higher in the FHF group. Lymphocyte proliferative response to rORF2p was comparable in both groups. As compared to AVH, significantly higher levels of both Th1 (IFN-gamma, IL-2 and TNF-alpha) and Th2 (IL-10) cytokines were recorded in FHF patients. Analysis of sequential samples differentiated FHF recovered and fatal patients with respect to IFN-gamma and IL-12. CONCLUSION: The results document increased Th1/Th2 responses and anti-HEV titers in FHF patients that warrant in-depth immunological studies.

Influenza B in a temperate region of northern India 2010-2016: co-circulation of the two lineages with northern hemispherical seasonality.

Scant data exist about the epidemiology of influenza B in India. We set out to address the epidemiology of influenza B in a temperate region of northern India from 2010 to 2016. Outpatient and inpatient surveillance was conducted in patients presenting with acute respiratory infection in a northern Indian hospital from September 2010 till April 2016. After recording clinical data, combined nasal/throat swabs were collected and tested for influenza viruses by real time RT-PCR. Influenza A viruses were further subtyped into A/H3N2 and A/H1N1 whereas influenza B were differentiated into B/Yamagata and B/Victoria. Virus isolation, haemaggglutination inhibition testing, sequencing and phylogenetic analysis was carried out on representative samples. Of the 6879 recruited cases, influenza B was detected in 299 (4.3%). The patients presented with respiratory symptoms of varying duration; cough, fever and nasal discharge being the most common. The peaking of the activity of the circulation showed a correlation with the onset of the winter with reduced temperatures and high dry humidity. B/Victoria lineage was detected in 35.4% (n = 106/299) whereas 53.8% (n = 161/299) were B/Yamagata. The circulation in each season was dominated by one lineage which correlated with the vaccine strain, but up to 37% consisted of a different lineage. We conclude that Influenza B exhibits a northern hemispherical seasonality in temperate northern India with co-circulation of the 2 lineages of influenza B. These findings have relevance for vaccine effectiveness and argue for vaccination with a quadrivalent influenza vaccine.

Molecular detection and characterization of Influenza 'C' viruses from western India.

Since 2003, India has had a well-established influenza surveillance network, though Influenza C virus was not the focus of study. We therefore retrospectively analyzed clinical samples from Pune, western India collected during January 2009 to August 2015, by real-time RT-PCR. Three of 2530 samples of patients with influenza-like illness (ILI) or severe acute respiratory illness (SARI) showed positivity for Influenza C virus infection, while 105 and 31 samples were positive for Influenza A and B viruses respectively. Influenza C viruses were successfully isolated using the embryonated egg system and whole genomes were sequenced and analyzed phylogenetically. HE gene-based phylogeny showed that two viruses C/India/P119564/2011 and C/India P121719/2012 clustered with the C/Sao Paulo/378/82 (SP82) lineage, whereas C/India/P135047/2013 clustered with the C/Kanagawa/1/76 (KA76) lineage. The internal gene of these viruses grouped in two lineages. The PB1, PB2, M and NS genes of the study viruses grouped with C/Yamagata/26/81 (YA81), while the P3 (PA) and NP genes grouped with C/Mississippi/80 (MS80). Bayesian clock studies conclude that the Indian strains may have emerged through multiple reassortment events.

Performance of rapid influenza diagnostic tests (QuickVue) for influenza A and B Infection in India.

BACKGROUND: Rapid point-of-care (POC) tests provide an economical alternative for rapid diagnosis and treatment of influenza, especially in public health emergency situations. OBJECTIVES: To test the performance of a rapid influenza diagnostic test, QuickVue (Quidel) as a POC test against a real-time polymerase chain reaction (RT-PCR) assay for detection of influenza A and B in a developing country setting. STUDY DESIGN: In a prospective observational design, 600 patients with influenza-like illness (ILI) or with severe acute respiratory illness (SARI) who were referred to the Influenza Clinic of a tertiary care hospital in Srinagar, India from September 2012 to April 2013, were enrolled for diagnostic testing for influenza using QuickVue or RT-PCR. All influenza A-positive patients by RT-PCR were further subtyped using primers and probes for A/H1pdm09 and A/H3. RESULTS: Of the 600 patients, 186 tested positive for influenza A or B by RT-PCR (90 A/H1N1pdm09, 7 A/H3 and 89 influenza B), whereas only 43 tested positive for influenza (influenza A=22 and influenza B=21) by QuickVue. Thus, the sensitivity of the QuickVue was only 23% (95% confidence interval, CI: 17.3-29.8) and specificity was 100% (95% CI: 99.1-100) with a positive predictive value (PPV) of 100% (95% CI 91.8-100) and a negative predictive value (NPV) of 74.3% (95% CI: 70.5-77.9) as compared to RT-PCR. CONCLUSIONS: The high specificity of QuickVue suggest that this POC test can be a useful tool for patient management or triaging during a public health crisis but a low sensitivity suggests that a negative test result need to be further tested using RT-PCR.

Total synthesis of heterocyclic steroids, Part IV. Mass spectrometry in stereochemical determination of the C/D ring fusion geometry in some heteroaromatic steroids.

The mass spectral fragmentation pattern and the relative intensities of the fragment peaks of the D ring in some 8-dehydroheteroaromatic steroids were studied. Characteristic differences for the cis and trans isomers were ascertained and were used for the determination of the stereochemistry of thiasteroids of which only one isomer was obtained.

Detection of N-nitrosamines in the saliva of habitual chewers of tobacco.

The saliva of habitual chewers of tobacco and of non- chewers was examined using high-performance liquid chromatography and gas-liquid chromatography-thermal energy analysis. N-Nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosonornicotine and N- nitrosohydroxyproline were detected in the saliva from the tobacco chewers . These N-nitroso compounds are probably leached from the tobacco itself and/or are formed in situ by the nitrosation of tobacco alkaloids as well as that of secondary amines. No N-nitroso compounds were detected in the saliva of non- chewers .

Seroepidemiology of influenza in Pune, India.

BACKGROUND & OBJECTIVES: Influenza viruses cause frequent epidemics and periodic pandemics throughout the world due to antigenic variations. Serological data can be useful to determine the disease burden and population immunity and for predicting the likelihood of occurrence and potential severity of subsequent epidemics. We undertook a serological analysis of antibodies against ten influenza virus strains in Pune, India. METHODS: Haemagglutination inhibition (HI) test was done on 619 sera collected between 1997-99 during an age-stratified serosurvey in Pune, India against 10 strains of influenza virus. Overall prevalence and spectrum of HI antibodies against these strains was determined. RESULTS: Antibodies to at least one influenza virus strain was seen in 62 per cent (116/188) of the sera from individuals in the age group 5-15 yr, 77 per cent (85/111) in sera from 16-25 yr, 78 per cent (93/119) from 26-35 yr, 84 per cent (77/92) from 36-45 yr and 93 per cent (101/109) in sera from individuals aged > 45 yr. The antibody spectrum progressively increased with age. Antibodies to the pandemic strain A(H2N2) were absent in the age groups < 25 yr. INTERPRETATION & CONCLUSION: The results indicate that influenza virus infection occurs in a large proportion of individuals in our community and may be responsible for a considerable amount of morbidity and mortality. The study also demonstrates the absence of antibody to A/Singapore/1/57 (H2N2) strain in younger persons < 25 yr of age. The potential of its reintroduction cannot be ruled out as H2 variants are circulating in wild birds and population immunity in humans is decreasing.

Hepatitis B infection among dental personnel in Pune & Bombay (India).

To assess the risk of hepatitis B infection among dental personnel, serum samples were collected from dentists of Pune and students, staff, auxiliary staff and class D staff of a dental college in Bombay. Dentists (32.02%), dental auxiliary staff (35.89%), clinical assistants and post-graduate students (19.56%) were found to have significantly higher prevalence of HBV infection as compared to undergraduate dental students (3.94%). The prevalence of HBV infection was high among the dentists as compared to voluntary donors. A positive linear association was observed in the positivity of HBV seromarkers with increasing age and number of years spent by the workers in the dental environment. The rate of increase in HBV seropositivity with age was higher (P less than 0.05) among dental personnel when compared to voluntary donors. Vaccination against hepatitis B is recommended for all the dental students before they start their clinical phase and for susceptible dentists and dental auxillary staff.

Prevalence of anti-HCV antibodies in western India.

Nearly 2000 serum samples collected from different risk groups from Pune and Bombay metropolitan areas were tested for antibodies to hepatitis C virus (anti-HCV) by Recombinant Immunoblot Assay-3 (RIBA-3). Patients undergoing haemodialysis showed 24.5 per cent seropositivity whereas 5.7 and 5.3 per cent of multiply transfused patients (>2 units) and chronic liver disease patients respectively were anti-HCV positive. Leprosy patients had almost 0.7 per cent seropositivity. In other risk groups the positivity rate was nil. In normal population only one out of 830 persons had anti-HCV antibodies. It is therefore apparent that the prevalence of hepatitis C virus (HCV) in western India is not high. However, special care needs to be taken for dialysis patients. As none of the 430 pregnant women and 86 children below the age of 5 yr were anti-HCV positive, vertical mode of HCV transmission seems to be negligible.

Exposure of Indian children to hepatitis A virus & vaccination age.

It is known that 90 per cent of children in India are exposed to hepatitis A virus (HAV) by the age of six years. The aim of the study was to determine when in early childhood maximum HAV infections take place and to deduce an appropriate age for vaccination against HAV. Blood samples of 499 children between the ages of three days and six years were collected and tested for the presence of antibodies against hepatitis A. A statistically significant negative correlation between IgG anti-HAV and age was observed (P < 0.01) up to 11.67 months when IgG anti-HAV positivity was found to be minimum (9.25%). Subsequently a significant positive correlation was noted (P < 0.01). Exposure to HAV was 28.9 per cent soon after the waning of maternal antibodies in the 13-15 month age group which increased to 52.5 per cent by two years of age and 90.9 per cent by 6 yr. It is concluded that in addition to other preventive measures, if children in India are to be vaccinated against hepatitis A they should be immunised against HAV by 9-10 months of age when the maternal antibodies disappear.

Hepatitis E virus infection in pregnant rhesus monkeys.

Ten non-pregnant female monkeys and four pregnant monkeys (all Macaca mulatta) in the last third of their gestation period were infected intravenously with the stool sample of a patient with hepatitis E virus infection (immuno-electronmicroscopy positive for hepatitis E virus). Four more non-pregnant monkeys were inoculated with a lower dose (less number of virus particles by IEM) of a stool sample collected on a different day from the same patient. The average incubation period as evidenced by the rise of serum alanine transferase in the non-pregnant monkeys, was 36.4 +/- 4.9 days. The dose of the virus did not affect the incubation period. Two of the pregnant monkeys had incubation periods of 9 and 13 days respectively. They delivered healthy babies on 40th and 53rd day respectively after inoculation. At the age of 11 months, both babies were negative for anti-HEV antibodies. One monkey which delivered a healthy baby on the 2nd day after inoculation had incubation period of 36 days. The baby of this monkey was anti-HEV positive at the age of 11 months. The incubation period was 41 days in the fourth monkey which delivered a macerated foetus on the 36th day after infection. No fatality was recorded in the infected monkeys. Bile samples collected from all monkeys showed strong signals in nested polymerase chain reaction (PCR). It seems that the incubation period in pregnant monkeys was determined by the state of pregnancy.

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.

Use of filter paper disks for hepatitis A surveillance.

BACKGROUND: Venous blood collection is a cumbersome and uncomfortable procedure during hepatitis A surveillance. Collection of capillary blood by finger prick is an alternative method. AIM: To evaluate the reactivity of capillary blood/anti-hepatitis A virus (HAV) IgG stored on filter paper disks for detection of anti-HAV antibody. METHODS: Venous blood specimens were collected from healthy individuals. Simultaneous capillary blood specimens obtained by finger prick were stored on filter paper disks. A reference standard of anti-HAV IgG in known concentrations was spotted on filter paper disks. The reactivities of anti-HAV IgG and capillary blood specimens eluted from filter paper disks were tested by blocking ELISA for detection of anti-HAV antibody. The results were evaluated by comparing optical density (OD) and neutralization values with those obtained for WHO anti-HAV IgG stored in liquid phase and homologous venous blood specimens, respectively. RESULTS: Among both venous and capillary-blood specimens stored for 10 days, percent neutralization shown by the same 46 specimens was > 50 and that of the same 3 specimens was < 50, indicating anti-HAV positivity and negativity, respectively. There was significant correlation between the OD values displayed by anti-HAV IgG from liquid phase and that eluted from filter paper disk (p < 0.01). Sixteen serum specimens stored for a period of 2 months showed results similar to those of the corresponding filter paper disk elutes. CONCLUSION: Use of filter paper disks could be a suitable choice for pre- and post-immunization collection of blood specimens during hepatitis A surveillance.

Ten-year serological follow up of hepatitis B vaccine recipients.

AIM: To determine long-term persistence of antibodies to hepatitis B surface antigen (anti-HBs) after vaccination against hepatitis B. METHODS: Thirty-four laboratory workers received hepatitis B vaccine in 1989 in a 0-1-6 month vaccination schedule. Group A (n = 16) received a booster at 3 years after vaccination whereas Group B (n = 18) did not. Anti-HBs was quantitated at 1 month and 1, 2, 3, 5, 6 and 8 years post-vaccination. RESULTS: At eight-year follow up, 10 of 15 subjects in Group A and 3 of 16 in Group B had protective levels of anti-HBs; in addition, two and four subjects, respectively, had detectable anti-HBs though below protective levels. At ten years, 9/15 and 3/16 were anti-HBs positive in Groups A and B, respectively. One subject in each group had rise in anti-HBs titer at 6-year follow up but both of them tested negative for IgG antibodies to hepatitis B core antigen (anti-HBc). A booster dose at 10 years to anti-HBs negative subjects led to an anamestic response in 3/4 and 8/10 persons in Groups A and B, respectively. CONCLUSION: Immunological memory after vaccination against hepatitis B is maintained for at least 10 years.