Subhash Medhi

Role of polymorphic N-acetyl transferase2 and cytochrome P4502E1 gene in antituberculosis treatment-induced hepatitis.

Background and Aim:  Antituberculosis drugs, isoniazid and rifampicin, in combination, are known to develop drug‐induced hepatotoxicity (DIH). A higher risk of DIH during antituberculosis treatment (ATT) has been reported in the Indian subcontinent compared to its Western counterparts. The role of genetic factors in a higher incidence of ATT hepatotoxicity in the Indian population is still unclear. The present study was aimed at investigating the role of the N‐acetyltransferase2 (NAT2) and cytochrome P4502E1 (CYP2E1) gene polymorphisms in ATT hepatotoxicity.

Evidence of extrahepatic replication of hepatitis E virus in human placenta.

The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with high fetal mortality. Based on this evidence and in the absence of reports on HEV replication in extrahepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative-strand-specific reverse transcriptase PCR. Viral load was estimated by real-time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in blood samples. Positive staining of placental tissue sections with HEV antibody against the viral structural protein ORF3 was observed. HEV replication in placenta also correlated with fetal and maternal mortality in ALF patients. It is demonstrated for the first time that HEV replication occurs in human placenta and that placenta may be a site of extrahepatic replication of HEV in humans.

Diagnostic utility of hepatitis C virus core antigen in hemodialysis patients

BACKGROUND Patients undergoing hemodialysis are at high risk for Hepatitis C virus infection. Anti-HCV antibody detection is widely used for screening this infection but is not sensitive for window period detection. An ELISA to detect the HCV Core Antigen has recently become available. OBJECTIVES To investigate the utility of the HCV core Antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with 3rd generation ELISA validated by real-time PCR. METHODS Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and Total HCVcAg was determined by third generation ELISA kits. HCV RNA was determined using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and sensitivity of the two assays was confirmed by estimating viral load using real-time PCR. RESULTS Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, which is statistically significant (P<0.05). All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of 49,258+/-28,682 copies/mL were detected in HCVcAg positive cases in comparison to 239,383+/-107,805 copies/mL in the only anti-HCV positive group (P<0.001). False negative cases for HCVcAg assay accounted for 2/250 (0.8%) in which the viral load was 306+/-461 copies/mL which was significantly lower in comparison to HCVcAg positive group (P<0.001, t-test=9.982). CONCLUSIONS Total HCVcAg ELISA is an accurate serological marker for early identification of HCV infection, than is possible by currently used serological assay. It will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. It is both a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.

Hepatitis C virus related hepatocellular carcinoma: a case control study from India.

Infection with hepatitis C virus (HCV) is a major risk factor for hepatocellular carcinoma (HCC) in India. The study was designed to investigate the clinical and molecular profiles of HCV related HCC cases in Indian patients. In a prospective study, 68 HCV related HCC, 55 HCV related chronic hepatitis, and 68 HCV related patients with cirrhosis were included. Glutathione S‐transferase gene polymorphism was analyzed in all the cases. The sex ratios were 5.18:1, 1.39:1, and 0.83:1 with mean age of 50.57 ± 12.47, 39.41 ± 13.34, and 46.08 ± 15.06 years, respectively, in three groups. Amongst the HCV related HCC cases seen in India, 49.2% (30 out of 68) were with Okuda stage I while 34.4% (21 out of 68) cases were classified as stage II. Older age, poor standards of living, HCV genotype 4, smoking, and null genotypes of GST were the risk factors associated significantly with the development of HCC. In 55.9% cases (38 out of 68) the size of the tumor was ≥5 cm while in 38.2% cases (26 out of 68) the size was between 2 and 5 cm, indicating an advanced stage of the disease at presentation. J. Med. Virol. 84: 1009–1017, 2012.

Viral Genotypes and Associated Risk Factors of Hepatocellular Carcinoma in India

Objective This study aims to investigate the etiological relationship among hepatitis B virus (HBV), hepatitis C virus (HCV), and alcohol as risk factors in a cohort of hepatocellular carcinoma (HCC) patients from India. The clinical and biochemical profiles and tumor characteristics in the HCC cases were also evaluated. Methods A total of 357 consecutive cases of HCC fulfilling the diagnostic criteria from the Barcelona–2000 EASL conference were included in the study. The blood samples were evaluated for serological evidence of HBV and HCV infection, viral load, and genotypes using serological tests, reverse transcription-polymerase chain reaction, and restriction fragment length polymorphism. Results The male/female ratio for the HCC cases was 5.87:1. Majority of the HCC patients (33.9%) were 50 to 59 years of age, with a mean age of 4±13.23 years. More than half the cases (60.8%) had underlying cirrhosis at presentation. Among the HCC patients, 68.9% were HBV related, 21.3% were HCV related, 18.8% were alcoholic, and 18.2% were of cryptogenic origin. The presence of any marker positive for HBV increased the risk for developing HCC by almost 27 times [OR: 27.33; (12.87–60.0)]. An increased risk of 10.6 times was observed for HCC development for cases positive for any HCV marker [OR: 10.55; (3.13–42.73)]. Heavy alcohol consumption along with HCV RNA positivity in cirrhotic patients was found to be a risk for developing HCC by 3 folds [OR: 3.17; (0.37–70.71)]. Conclusions Patients of chronic HBV infection followed by chronic HCV infection were at higher risk of developing HCC in India. Chronic alcohol consumption was found to be a risk factor in cirrhotic cases only when it was associated with HCV RNA positivity. Most of the patients had a large tumor size (>5 cm) with multiple liver nodules, indicating an advanced stage of the disease thus making curative therapies difficult.

Prevalence of Hepatitis B Virus Genotype D in Precore Mutants Among Chronic Liver Disease Patients from New Delhi, India

Hepatitis B is one of the most important causes of chronic viral hepatitis world wide. Mutations in the precore region of the hepatitis B virus (HBV) genome are frequently found in hepatitis B envelope antigen-negative cases. Data from India on the HBV genotype-associated distribution of precore mutations are limited. Our objective in this study was to genotype and detect the precore mutant with a point mutation from G to A at nucleotide 1896 using ligase chain reaction (LCR) and direct sequencing. A total of 115 cases of chronic liver disease were screened. The cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test for HBV infection, which includes HBsAg, anti HBcIgG, HBeAg using commercially available ELISA kits. The cases, which were HBeAg+, HBeAg–, and HBV DNA+, were subjected to LCR and confirmed by direct sequencing. Of 115 chronic liver disease cases, 50 (43.5%) cases were HBV DNA positive. All cases were subjected to LCR; 11 (22%) cases confirmed the presence of precore mutants, while the remaining 39 (78%) were classified as the wild form of the virus. HBV genotyping by direct sequencing revealed that genotype D was predominant in both wild and mutant forms of the virus. We conclude that the HBV genotype distribution was not significantly different between precore mutants and the wild form of the virus (P>0.05). North Indian patients with genotype D were more likely to have persistent HBV infection with precore mutants. HBV genotypes correlate with the clinical outcome of chronic HBV infection.

Genetic variants of heat shock protein A1L2437 and A1B1267 as possible risk factors for hepatocellular carcinoma in India.

To study the role of heat shock protein A1L (HSPA1L) and A1B (HSPA1B) polymorphisms and subsequent risk of hepatocellular carcinoma (HCC) in India. Subjects enrolled included 185 cases of HCC, 182 cases of chronic hepatitis (CH) and 200 healthy controls. Genomic DNA was typed for HSPA1L2437 and HSPA1B1267 SNP using polymerase chain reaction with restriction fragment length polymorphism. Other risk factors were also analysed. Hepatitis B virus (HBV) infection, older age >35 years and high aflatoxin level in urine increased the risk of HCC. The frequencies of HSPA1L BB genotype and B allele in HCC were more than in CH [odds ratio (OR): 9.83; P = 0.000], but also in HBV‐related HCC than Chronic Hepatitis B (CHB) [OR: 3.44; P = 0.004] and HCV‐related HCC compared to CHC [OR: 6.32; P = 0.010]. The frequency of HSPA1B genotype in the homozygous state was more in CH [OR: 6.01; P = 0.001] and is a good marker to predict the risk of HCV‐related CH (CHC) compared to controls. HCV‐related HCC has a higher frequency of the B allele of HSPA1B than healthy controls [OR: 3.95; P = 0.000] and CHC [OR: 2.35; P = 0.000], respectively. The frequencies increased further significantly in CHC compared to healthy controls [OR: 9.26; P = 0.000]. The risk for the development of CH and HCC compared to healthy controls irrespective of the aetiology was significant in terms of the HSPA1B marker than HSPA1L in the Indian population.

Molecular epidemiology of hepatitis A virus infection in Northeast India.

The present study was undertaken to screen the molecular epidemiology of Hepatitis A virus (HAV) in Northeast India (NEI) who are ethnically distinct, tribal dominated and of lower socio‐economic status with almost no information available from NEI on these aspects. Briefly, 3 ml blood was collected from 324 random liver disease cases with jaundice, receiving care at Central Hospital, N.F. Railway, Guwahati, Assam with informed consent. The patients detected with HAV‐IgM positive status were included and were stratified as acute viral hepatitis (AVH) and fulminant hepatitis (FHF) based on clinical profile. Viral RNA was isolated and HAV‐RNA was detected by Real‐time PCR using primers for the VP3–VP1 region. HAV genotyping was studied by PCR‐direct sequencing‐phylogenetic analysis approach using the VP1/2A region of HAV isolates. Statistical analysis was performed using SPSS13.0 software. A total of 69 cases were HAV infected with two HBV co‐infected cases (n = 69 + 2 = 71), 62 cases and two co‐infected cases were AVH and others were FHF cases. HAV infection was predominant in especially in the young and adult age group. HAV‐RNA was detected in 28 cases, out of which 19 cases could be genotyped (12 AVH, 7 FHF); which showed the prevalence of genotype IIIA or IA only. Although HAV genotype IIIA was the major genotype in both the AVH (10/12, 83.33%) and FHF (5/7, 71.43%) group, but the difference in distribution of genotypes in AVH and FHF cases was statistically non‐significant (P = 0.550). HAV genotype IIIA is associated with the majority of HAV infected cases and severity in NEI. J. Med. Virol. 87:1218–1224, 2015.

Investigations on implementation of e-ATM Web Services based on .NET technique

Web service is accepted by all communities of services due to its interoperability, flexibility of design and architecture. Improving performance is the key research point for developers and researchers. We propose to design, develop and implement a prototype research electronic automated teller machine web service to study the loads, performance and scalability of the service. The service is based on Visual Studio .NET framework. It is developed by using C# as programming language, Internet Information Service (Version: 2005) as web server and, Microsoft Structured Query Language as database server. To study its different aspects the service has been tested by deploying it on Mercury LoadRunner (Version 8.1). In this paper we will present the architecture, testing procedure, results of performance testing as well as results of statistical analysis on recorded data of the service.

Alteration in the expression of MGMT and RUNX3 due to non-CpG promoter methylation and their correlation with different risk factors in esophageal cancer patients

Promoter methylation reflects in the inactivation of different genes like O6-methylguanine-DNA methyltransferase DNA repair gene and runt-related transcription factor 3, a known tumor suppressor gene in various cancers such as esophageal cancer. The promoter methylation was evaluated for O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in CpG, CHH, and CHG context (where H is A, T, or C) by next-generation sequencing. The methylation status was correlated with quantitative messenger RNA expression. In addition, messenger RNA expression was correlated with different risk factors like tobacco, alcohol, betel nut consumption, and smoking habit. CpG methylation of O6-methylguanine-DNA methyltransferase promoter had a positive association in the development of esophageal cancer (p < 0.05), whereas runt-related transcription factor 3 promoter methylation showed no significant association (p = 1.0) to develop esophageal cancer. However, the non-CpG methylation, CHH, and CHG were significantly correlated with O6-methylguanine-DNA methyltransferase (p < 0.05) and runt-related transcription factor 3 (p < 0.05) promoters in the development of esophageal cancer. The number of cytosine converted to thymine (C→T) in O6-methylguanine-DNA methyltransferase promoter showed a significant correlation between cases and controls (p < 0.05), but in runt-related transcription factor 3 no such significant correlation was observed. Besides, messenger RNA expression was found to be significantly correlated with promoter hypermethylation of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in the context of CHG and CHH (p < 0.05). The CpG hypermethylation in O6-methylguanine-DNA methyltransferase showed positive (p < 0.05) association, whereas in runt-related transcription factor 3, it showed contrasting negative association (p = 0.23) with their messenger RNA expression. Tobacco, betel nut consumption, and smoking habits were associated with altered messenger RNA expression of O6-methylguanine-DNA methyltransferase (p < 0.05) and betel nut consumption and smoking habits were associated with runt-related transcription factor 3 (p < 0.05). There was no significant association between messenger RNA expression of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 with alcohol consumption (p = 0.32 and p = 0.15). In conclusion, our results suggest that an aberrant messenger RNA expression may be the outcome of CpG, CHG, and CHH methylation in O6-methylguanine-DNA methyltransferase, whereas outcome of CHG and CHH methylation in runt-related transcription factor 3 promoters along with risk factors such as consumption of tobacco, betel nut, and smoking habits in esophageal cancer from Northeast India.