Sadras Panchatcharam Thyagarajan

A reliable and inexpensive EasyCD4 assay for monitoring HIV-infected individuals in resource-limited settings.

Summary: Serial measurements of absolute CD4+ T-lymphocyte counts are required to initiate and gauge response to therapy and monitor disease progression. Hence, there is an urgent need to evaluate the accuracy and validity of low-cost CD4+ T-cell count assays. Tripotassium EDTA blood specimens from HIV-infected individuals were studied using a novel flow cytometric assay (EasyCD4 assay; Guava Technologies, Hayward, CA) in comparison with standard flow cytometry (FACSCount; Becton Dickinson Immunocytometry Systems, San Jose, CA). The sensitivity, specificity value by EasyCD4 assay in enumerating absolute CD4+ T-cell counts of less than 200 cells/&mgr;L were 95% and 100%, respectively. Bland-Altman analysis showed close agreement, with the EasyCD4 assay yielding CD4+ T-cell counts a mean difference of −26 cells/&mgr;L (95% confidence interval, −96 to 44 cells/&mgr;L) higher than by flow cytometry. Our data suggest that EasyCD4 assay could be a useful alternative assay to conventional flow cytometry, may be appropriate for use in resource-limited settings.

Genital chlamydial infection in STD patients: its relation to HIV infection.

In the present report, we have analysed C.trachomatis infection and HIV positivity among patients (n-143) who attended the STD clinic at the Institute of STDs, Government General Hospital, Chennai. HIV positivity rate was significantly high among those with chlamydial infection than in those without chlamydial infection (29.5% (13/44) vs. 11.1% (11/99); p<0.05). The results of the present study suggest the association between C.trachomatis and HIV infections and reinforce the need for routine screening for C.trachomatis as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in India.

Herpes simplex keratitis in South India : Clinico-virological correlation

PURPOSE A retrospective cross-section study to analyze the prevalence of herpes simplex virus-induced keratitis (HSK) among 3,000 patients attending a corneal clinic in South India between 1995 and 1997, and to evaluate laboratory techniques for detecting HSK. METHODS The clinico-virological correlation was studied using herpes simplex virus (HSV) isolation on the Vero cell line, HSV-specific antigen detection by indirect immunofluorescence (IF) microscopy, and serum anti-HSV IgG quantitation, IgM estimation, and tear secretory IgA (sIgA) detection by ELISA. OBSERVATIONS HSK had a prevalence of 7.8% (234 patients) in this study. A virological correlation could be obtained in 44.4% of the cases that had epithelial manifestations and in 14.8% of the cases that had only stromal disease. In 161 cases where both culture and IF microscopy were used, IF detected 27 cases (26.8%) more than cell culture. The difference in sensitivity between cell culture and IF was found to be statistically significant (McNemars test, P < .05). An elevation in IgG titer was seen in 17 (30.4%) cases. IgM was detected in only 2 cases of the 62 (3.2%) analyzed. Of the 138 cases analyzed, sIgA was positive in 28 (20.3%) cases. A proved diagnosis could be made in 58% of cases when the specimen was collected during the first week after disease onset, and in only 5% when the time interval increased to 4 weeks. CONCLUSIONS HSV antigen detection by indirect IF is a rapid and sensitive diagnostic tool for HSK. Tear secretory IgA (sIgA) is a specific marker for acute herpetic keratitis, and the detection of HSV-specific tear sIgA is a valuable adjunct to virus isolation and antigen detection in the laboratory diagnosis of HSK. For a successful diagnosis, the specimen should be collected as soon as possible after HSK onset.

Alpha-fetoprotein as a tumor marker in hepatocellular carcinoma: investigations in south Indian subjects with hepatotropic virus and aflatoxin etiologies

OBJECTIVES The prevalence of hepatitis B virus (HBV) is reportedly the main cause of hepatocellular carcinoma (HCC) in India, where hepatitis C virus (HCV)-associated HCC is believed to be relatively less prevalent. We verified the usefulness of alpha-fetoprotein (AFP) as a tumor marker and analyzed the influence of viral etiology on AFP levels in HCC. METHODS Of a total of 1012 cases with liver disease, 202 were investigated for the presence of AFP (142 HCC cases, 30 cirrhosis cases, and 30 chronic liver disease (CLD) cases). In addition, serum samples from 30 healthy patients, 30 hepatitis B surface antigen (HBsAg) carriers, and 30 acute viral hepatitis cases were included as controls. AFP was quantitatively determined using a commercial ELISA (Quorum Diagnostics, Canada). Out of the 142 HCC cases screened for AFP, aflatoxin B1 (AFB1) detection was carried out in 38 HCC cases using an in-house immunoperoxidase test. RESULTS In HBV and HCV co-infected HCC cases, the AFP positivity was 85.7%. In HBV alone-associated HCC, the positivity was 62.9%, and 54.5% of AFB1 positive HCC cases showed AFP positivity. In HBV and HCV negative HCC cases, the positivity was 20.5%, and in HCV-associated HCC it was 17.6%. The HBV/HCV co-infected group and HBV alone positive HCC cases had significantly elevated levels of AFP. When AFP positivity was analyzed based on the marker profile of HBV, 89.7% of AFP positive cases were HBV-DNA positive. CONCLUSIONS The overall positivity pattern of AFP in HCC does indicate that higher levels of AFP are observed with hepatitis virus positivity, especially with HBV. Further studies must be carried out to correlate the serum levels of AFP with the size, number, and degree of differentiation of HCC nodules.

Genital syndromes and syndromic management of vaginal discharge in a community setting

The objective of the study was to determine the community prevalence of genital syndromes in women and evaluate the syndromic management of vaginal discharge in this setting. A representative sample for the state of Tamilnadu was chosen using probability proportional to size cluster technique. Thirty clusters were selected from three districts. Demographic, sexual behaviour, risk factors, clinical and laboratory data were collected from the selected population using a structured questionnaire. Direct smear examination for Trichomonas vaginalis, culture for Neisseria gonorrhoeae and Haemophilus ducreyi, serological tests for syphilis (RPR and TPHA), hepatitis B (Hbs Ag ELISA), IgM and IgG antibodies to HSV2 (Novum diagnostics, Germany) and PCR test for detection of C. trachomatis from urine were done. There were 1157 women in the selected population. On examination, vaginal discharge was the most common genital syndrome (38.4%). The sensitivity, specificity, positive and negative predictive value of vaginal discharge as a marker for STD in women was found to be 43.3%, 61.6%, 10.7% and 91.1%, respectively. We concluded that treatment on the basis of syndromic management would result in over-treatment of 90% of women with vaginal discharge.

The prevalence of hepatitis B virus and hepatitis C virus infection among patients with chronic liver disease in south India.

OBJECTIVE Determining the identity of hepatitis C virus (HCV) genotypes in liver disease has key implications for ascertaining the duration of antiviral therapy and disease prognosis. We investigated the presence of various genotypes of HCV among 69 chronic liver diseased (CLD) patients with chronic HCV infection. METHODS Sixty-nine consecutive subjects with underlying chronic hepatitis (n=28), cirrhosis (n=35), and hepatocellular carcinoma (n=6), diagnosed by clinical, biochemical, and histological means, were studied. Hepatitis B virus (HBV) and HCV diagnostic markers were used. HCV-RNA was extracted from sera of HCV-infected subjects and subsequently the HCV genotypes were determined using a commercial line probe assay (Inno-LiPA HCV II). RESULTS Of the 69 CLD cases screened for possible markers of HBV and HCV infection, 39 (57%) were positive for HBV and 30 (43%) were HCV infected. The overall HCV-RNA positivity was 77% (23/30). Of these, the majority were genotype 1b (13/23, 57%), followed by 1a (6/23, 26%), mixed genotypes 3 and 4(3/23, 13%), and mixed pattern of 1a, 1b, and 4 (1/23, 4.3%). The genotype 1b infected subjects demonstrated significantly elevated transaminase (ALT) levels (p<0.05) as compared with the other non-1b HCV genotypes. CONCLUSIONS The predominance of HCV genotype 1b among CLD patients could pose a major challenge for the efficient management of HCV disease and the development of effective therapeutic interventions in peninsular India.

Bacterial vaginosis in female sex workers in Chennai, India.

Bacterial vaginosis (BV) causes obstetric and gynaecological complications and non-chlamydial/non-gonococcal pelvic inflammatory disease and has been shown to be associated with the risk of acquiring HIV and herpes simplex (HSV)-2 infections. This study investigated both the prevalence of BV and its association with STDs among 582 female sex workers living in Chennai, South India. Blood, vaginal and endocervical swabs were tested for HSV-2, HIV, Treponema pallidum, BV, Chlamydia trachomatis, Neisseria gonorrhoea and Trichomonas vaginalis. The vaginal swabs collected were Grams stained and analysed for BV by Nugents scoring criteria. Of the women studied, 45% (95% CI, 40.6-48.7) were positive, 39.5% (95% CI, 35.5-43.5) were negative and 16% (95% CI, 12.8-18.7) were intermediate for BV. Bacterial vaginosis positivity was directly related to concurrent infection with HSV-2 (RR 1.3, AR 12, P = 0.00), T vaginalis (RR 1.5, AR 10, P = 0.01) T. pallidum (RR 2.8, AR 16, P = 0.00) and HIV (RR 4.1, AR 52, P = 0.01). Future studies are needed to focus on the risk factors for BV.

Transmission of hepatitis C virus infection from asymptomatic mother to child in southern India

BACKGROUND Little information is available on the mother-to-child transmission of hepatitis C virus (HCV) in India, and no interventions to decrease transmission rates have been identified. Hence, we performed a long-term prospective study in infants born to HCV-positive mothers, with the aim of evaluating vertical transmission of HCV and correlated risks factors. METHODS Three thousand one hundred and fifteen healthy asymptomatic pregnant women were included in the study. We used third-generation (Murex anti-HCV) ELISA and HCV RNA reverse transcription PCR (RT-PCR) for screening, and the commercial line probe assay (Inno-LiPA) and direct sequencing HCV genotyping assays were performed to confirm the transmitted HCV genotypes. RESULTS Of the total 3115 healthy asymptomatic pregnant women, 18 (0.6%) were positive for anti-HCV. Of the 18 anti-HCV-positive women, eight (44.4%) were positive for HCV RNA RT-PCR. HCV transmission was observed in two of the eight babies born to eight HCV RNA-positive mothers who were followed up for 12 months. HCV genotyping of the mother/child pairs revealed the persistent presence of mixed genotypes 1a and 4 throughout the follow-up period. None of the non-viremic (HCV RNA-negative) mothers transmitted HCV infection to their baby. In our study approximately 25% of vertical/perinatal transmission of HCV was observed among HCV RNA-positive antenatal women. CONCLUSIONS This study is of importance as it is the first report from India of a successful attempt to analyze the rate of vertical/perinatal transmission of HCV from infected mothers to their children by a prospective longitudinal follow-up study, and to characterize the pattern of genotype(s) of HCV present in the infected mother/baby pairs, so as to confirm the source of HCV acquired by the newborn babies.

Acyclovir resistance in herpes simplex virus isolates from keratitis cases : An analysis from a developing country

Seven herpes simplex type‐1 (HSV‐1) isolates from herpes simplex keratitis (HSK) cases clinically resistant to acyclovir (ACV) were analyzed for the mechanism of ACV resistance in them. The purpose of the study was to focus the attention of ophthalmologists on the frequency of occurrence of ACV resistance in HSK and to characterize such a phenomenon. We employed in‐vitro plaque reduction assay, thymidine kinase assay, polymerase chain reaction, single‐strand confirmation polymorphism analysis and sequencing to detect any mutation(s) in thymidine kinase gene in this analytical study. Four of the seven HSV‐1 isolates proved ACV resistant by plaque reduction assay and three of them showed reduced thymidine kinase activity. Altered mobility pattern indicative of mutation within 335 base pair PCR product bracketing the suggested homopolymer mutational hotspot (7 Guanosine) was detected in 2 of these 3 isolates. DNA sequencing showed a deletion at nucleotide position 336 from the tk gene transcription start in both the isolates. This mutation has generated the first TGA stop codon 27 nucleotides downstream in the tk open reading frame. Our study also suggests the need of clinical/molecular surveillance of ACV resistance in HSV types in a given geographic location for better management of HSV infections.

Comparative efficacy of two dosages of recombinant hepatitis B vaccine in healthy adolescents in India.

Background: Inclusion of hepatitis B vaccine in the Universal Programme of Immunization of all Asian and African countries is hampered by the economic burden on the health budget because of the cost of hepatitis B vaccines. Here we evaluated the immunogenicity, safety, efficacy, and the persistence of antibody to hepatitis B surface antigen (anti-HBs) titers of a new and a low cost recombinant hepatitis B vaccine GeneVac B, with 2 different dosages in healthy adolescents in India. Methods: GeneVac-B, a recombinant hepatitis B vaccine (Serum Institute of India, Pune, India), was administered in 10 or 20 μg dose intramuscularly to 2 groups of 100 healthy school-going adolescents at 0-, 1-, and 6-month intervals, who were followed up for 1 year. Group I received 20 μg doses whereas Group II received 10 μg doses. Blood samples were collected 1 month after each dose and 1 year after the third dose. The anti-HBs titers were assayed using commercially available kits to assess the immunogenicity of the 2 dosage schedules. Safety studies were also carried out. Results: The geometric mean titer value of the anti-HBs titer 1 month after the third dose was 2629 (mlU/mL) in Group I and 1373 mlU/mL for Group II subjects. One year after the third dose, the persistence of anti-HBs in those who had received 20 μg was 2262 mlU/mL whereas it was 1039 mlU/mL in the group receiving 10 μg doses. All the subjects in both the groups were seroprotected at 1 year after vaccination. None of the vaccinees exhibited serious adverse reactions throughout the study period. Conclusions: The study demonstrated the immunogenicity of the recombinant hepatitis B vaccine, and confirms that the 0.5 mL (10 μg) dose of GeneVac B can be administered with satisfactory safety and immunogenicity to adolescents up to 19 years of age, reducing the cost to less than U.S.