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.

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 infl uenza, especially in public health emergency situations. Objectives: To test the performance of a rapid infl uenza diagnostic test, QuickVue (Quidel) as a POC test against a real-time polymerase chain reaction (RT-PCR) assay for detection of infl uenza A and B in a developing country setting. Study Design: In a prospective observational design, 600 patients with infl uenza-like illness (ILI) or with severe acute respiratory illness (SARI) who were referred to the Infl uenza Clinic of a tertiary care hospital in Srinagar, India from September 2012 to April 2013, were enrolled for diagnostic testing for infl uenza using QuickVue or RT-PCR. All infl uenza 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 infl uenza A or B by RT-PCR (90 A/ H1N1pdm09, 7 A/H3 and 89 infl uenza B), whereas only 43 tested positive for infl uenza (infl uenza A = 22 and infl uenza B = 21) by QuickVue. Thus, the sensitivity of the QuickVue was only 23% (95% confi dence interval, CI: 17.3-29.8) and specifi city 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 specifi city 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.

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 Mm2 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.

Efficacy of teriparatide in increasing bone mineral density in postmenopausal women with osteoporosis--an Indian experience.

BACKGROUND AND OBJECTIVE: Osteoporosis is emerging as a leading cause of substantial morbidity in India, particularly in postmenopausal women. Teriparatide (recombinant human parathyroid hormone [1-34]) increases bone formation and improves bone microarchitecture, thereby reducing the risk of fractures. This study was conducted to evaluate the efficacy of teriparatide in increasing bone mineral density (BMD) in postmenopausal women with osteoporosis. MATERIAL AND METHODS: A randomised, prospective, multicentre, open-label, controlled study was conducted on 82 postmenopausal women with established osteoporosis. Patients were randomly divided into control and teriparatide groups, each group consisting of 41 patients. All the patients were supplemented with 1000 mg of elemental calcium and 500 IU of vitamin D throughout the study period of 180 days. Besides, teriparatide group patients were administered teriparatide 20 microg daily subcutaneously. Lumbar spine, femoral neck and total hip BMD, bone mineral content (BMC) and bone area were measured by dual energy x-ray absorptiometry (DXA) at baseline and at the end of 6 months of treatment. Bone biomarkers, such as serum bone specific alkaline phosphatase (BSAP) and serum osteocalcin (OC), representing bone formation, and urinary deoxypyridinoline (DPD), representing bone resorption were assessed at baseline, and at 3 and 6 months of treatment. RESULTS: During the study period, 9 patients (11%) were lost to follow-up--6 in control group (7.3%) and 3 in teriparatide group (3.7%). There was an excellent compliance to both oral and injectable medication. The investigational product teriparatide was well tolerated and there were no serious adverse events. In addition, there were no significant differences between the groups in the incidence of adverse events. The percentage of increase in lumbar spine BMD, which is the primary endpoint, was significantly (P < 0.001) higher in teriparatide group compared to that in control group (6.58% vs. 1.06%). Further, teriparatide significantly increased percentage of change in lumbar spine T-score (P < 0.001), BMC (P < 0.001) and bone area (P < 0.028) compared to control group at 6 months. Administration of teriparatide resulted in a significant percentage of increase in all the bone biomarkers in teriparatide group compared to control group patients at 3 and 6 months over baseline, thereby showing that there was a significant increase in bone turnover in teriparatide group of patients. CONCLUSION: These results show that teriparatide is an effective and safe drug in increasing the BMD and therefore, teriparatide provides yet another new therapeutic option for reducing the risk management of osteoporosis in postmenopausal women (clinicaltrials.gov number, NCT00500409).

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 G virus infection in India: prevalence and phylogenetic analysis based on 5' non-coding region.

OBJECTIVES: To determine the prevalence of hepatitis G virus (HGV) infection in western India and to carry out phylogenetic analysis of HGV isolates. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect HGV RNA in serum samples obtained from paid plasma donors, patients with hemophilia and voluntary blood donors. Nine Indian and one Kenyan HGV RNA-positive samples were sequenced in the 5' non-coding region (5'-NCR). Phylogenetic analysis based on the comparison of a 101 nucleotide fragment from a large number of HGV isolates from 22 countries (including Indian and Kenyan sequences obtained during the present study) was carried out. RESULTS: HGV RNA positivity rates among paid plasma donors from a commercial plasmapheresis unit (7/43, 16.3%) and patients with hemophilia (5/44, 11.4%) were significantly higher than that in voluntary blood donors (0/51; p=0.003 and 0.019, respectively). Among patients with acute non-A to E hepatitis and fulminant hepatic failure, 1 of 50 and 1 of 28 were HGV RNA-positive, whereas 6 of 49 (12%) patients with chronic liver disease had circulating HGV RNA. All Indian isolates belonged to genotype 2, whereas the Kenyan isolate formed a distinct branch within genotype 1 consisting of African isolates. CONCLUSION: Our results suggest existence of parenteral transmission of HGV in the Indian population. HGV was not an important cause of acute non-A to E hepatitis or fulminant hepatic failure among the patients investigated. Genotype 2 seems to be the most prevalent genotype in western India.

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.

Epidemic hepatitis E: serological evidence for lack of intrafamilial spread.

INTRODUCTION: Hepatitis E presents as epidemic as well as sporadic disease. Fecal contamination of drinking water results in epidemics of hepatitis E. The extent of intrafamilial spread needs to be assessed employing serological assays. AIMS: To understand the dynamics of intrafamilial spread of the disease. METHODS: The study was conducted using blood samples collected during the 1988 and 1989 epidemics of viral hepatitis in Kudal and Atit villages of Maharashtra state; the epidemics were subsequently shown to be due to hepatitis E virus (HEV). The one-time collection carried out at the end of the Kudal epidemic was from 184 apparently healthy individuals irrespective of family history of jaundice during the epidemic. In the Atit epidemic, 153 family contacts of 49 IgM anti-HEV positive patients were bled. An additional 151 blood samples were collected from apparently healthy individuals irrespective of family history of jaundice during the epidemic. One month later, blood samples were collected from 64 of the 153 family contacts. Relevant history was recorded each time. All serum samples were tested for ALT levels and for IgM and IgG antibodies to hepatitis E virus employing ELISA. RESULTS: IgM anti-HEV positivity among persons with family history of jaundice was not different from those without such a history (8/62 [12.9%] and 11/122 [9%] at Kudal; 9/57 [15.8%] and 22/94 [23.4%] at Atit; p > 0.1). Excluding IgG anti-HEV positive samples from the analysis also yielded non-significant results. Of the 32 follow-up samples collected from family contacts without IgG or IgM antibodies to HEV in the initial blood sample, 31 remained IgM and IgG anti-HEV negative at the end of 1 month. One of the family contacts was found to be IgG anti-HEV positive in the second blood sample. The disease was not related to the index case. CONCLUSION: Intrafamilial spread of HEV is negligible.

Prevalence of hepatitis A antibodies in western Indian population: changing pattern.

This report pertains to a retrospective study conducted between 1983 and 1995 at three time points to evaluate the prevalence of hepatitis A virus (HAV) infection in the population of Bhor Taluk, situated in western India. Serum samples from children and adults were tested for anti-HAV antibodies using blocking ELISA test. There was a significant decrease in anti-HAV prevalence among children aged 5-10 years in 1995 (87.36%) as compared to that of 1983 (97.58%) and 1987 (96.48%). All individuals >11 years of age were seropositive for anti-HAV antibodies. Anti-HAV prevalence was similar in the users of well water, but was significantly reduced in individuals supplied with piped water in 1995 (88.61%) compared with that in 1983 (98.77%). A significant decrease in anti-HAV positivity was noted in children from Bhor Taluk as compared to children from Pune bled in 1992. These results underline the need for periodic surveillance of seroepidemiology of hepatitis A to determine the measures for prevention and control of the disease.

Insignificant prevalence of antibodies to hepatitis C in a rural area of western Maharashtra.

OBJECTIVE: To determine the age-specific seroprevalence of hepatitis C virus (HCV) in a rural population in Maharashtra. METHODS: 1054 serum samples collected from apparently healthy persons were tested by recombinant immunoblot assay for antibodies against HCV (anti-HCV). Anti-HCV positive samples were tested for HCV-RNA by nested reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: One man tested positive for anti-HCV; his sample was also HCV-RNA positive. CONCLUSIONS: HCV infection is infrequent in this rural area in Maharashtra.

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 A in day care centre.

A focal outbreak of hepatitis was detected in a day-care centre for children centrally located in Pune. The source of infection was suspected to be an 11-year-old child who probably got the infection from his school. Seven out of 15 children from day-care centre developed clinical hepatitis. Two cases of secondary infection were identified among the family contacts of infected children. Sera from all the nine sick children were positive for anti-hepatitis A virus-IgM antibodies. A stool sample from a case of secondary infection showed presence of HAV-RNA by RT-nested PCR. These findings proved that the outbreak was caused by hepatitis A virus.

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.

Menstrual irregularities and lactation failure may precede thyroid dysfunction or goitre.

Menstrual and reproductive history of 178 women referred to the thyroid clinic was compared with 49 healthy controls. Cases were classified as euthyroid, hypothyroid or hyperthyroid after clinical examination and after serum T3, T4, TSH measurements. Reproductive history was related chronologically to symptoms and signs of thyroid dysfunction. Only 31.8% of hypothyroid and 35.3% of hyperthyroid women had normal menstrual pattern in contrast with 56.3% of Euthyroid and 87.8% of healthy controls (p < 0.001). Reproductive failure (infertility, pregnancy wastage, failure of lactation) occurred in 37.5% of hypothyroid and 36.5% of hyperthyroid cases against 16.3% of euthyroid and 16.7% of healthy controls (p < 0.05). Interestingly, in 45% of cases with menstrual abnormality, the anomaly was antecedent to other clinical features by a variable period of two months to ten years. Reproductive failure and lactation failure also preceded thyroid dysfunction or goitre. Reproductive dysfunction may therefore be considered as one of the presenting symptoms of thyroid disorders in women, keeping in mind both menstrual irregularities and lactation failure may also arise from other common or idiopathic origins. Especially in women with menstrual irregularities in the perimenopausal age if thyroid dysfunction is detected, pharmacotherapy may be a superior alternative to surgical interventions like hysterectomy.

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.

Comparative immunogenicity of different hepatitis B vaccines among certain high risk groups in India.

The immunogenicity of three different commercially available, well established Hepatitis B vaccines was determined. Three groups of high risk individuals were administered the vaccine, viz, Albugam (20 micrograms), Engerix-B (20 micrograms) and vaccine produced by Cheil Sugar & Co. (3 micrograms) respectively in a schedule of 3 doses as recommended by the manufacturers. A fourth group was administered a combination of the Albugam (2 doses) and Engerix-B (1 dose) vaccine. The sera collected 2 months after the 3rd dose showed seroconversion rates to be 100 percent and 95.34 percent respectively in the group which received the Albugam and Engerix-B vaccines respectively. The group which received a combination of both, the seropositivity rate was also 100 percent. The Chiel Sugar vaccine gave a seroresponse of only 52.38 percent. Geometric mean titres among the groups which received Albugam vaccine (501.30) were comparable to those receiving the combination of Engerix-B and Albugam (442.28). Those who received all 3 doses of Engerix-B vaccine showed a significantly lower GMT as compared to the above tow groups (GMT--43.14, p < .001, P < .001 respectively). The GMT in the group that received Cheil Sugar Vaccine was 3.81. Seroresponse was found to be inversely proportionate to age.

Non A non B hepatitis epidemic in Rewa district of Madhya Pradesh.

An epidemic of viral hepatitis occurred at Rewa district of Madhya Pradesh during December 1989 through April 1990. A total of 302 cases were admitted to Gandhi Memorial Hospital. Few cases were reported from the adjacent rural areas. Twenty six of the 40 wards of the city were affected and 7 wards were most affected with attack rates ranging from 3-7 per cent. Seventy one per cent of the hospitalised cases were 15 to 35 years of age. Males constituted 72.2% of the cases. In 37 patients (12.2%) the illness had a fatal outcome. The cases fatality rate was 7.9% in males and 20.6% in females. There were leakages in water supply pipe lines at many places which ran parallel to or were laid across open gutters. The source of infection appeared to be water contaminated by sewage. Results of serological tests indicated a non-A, non-B hepatitis viral aetiology of the epidemic.

Klinefelter's syndrome with unelevated serum gonadotropin levels (a case report).

Two cases of Klinefelter's syndrome with 47 XXY karyotype associated with unelevated follicular stimulating hormone (FSH) and luteinizing hormone (LH) levels are reported. The literature is briefly reviewed.