A Model of Care Optimized for Marginalized Remote Population Unravels Migration Pattern in India.

BACKGROUND AND AIMS: Access to basic health needs remains a challenge for most of world's population. In this study, we developed a care model for preventive and disease-specific health care for an extremely remote and marginalized population in Arunachal Pradesh, the northeasternmost state of India. APPROACH AND RESULTS: We performed patient screenings, performed interviews, and obtained blood samples in remote villages of Arunachal Pradesh through a tablet-based data collection application, which was later synced to a cloud database for storage. Positive cases of hepatitis B virus (HBV) were confirmed and genotyped in our central laboratory. The blood tests performed included liver function tests, HBV serologies, and HBV genotyping. HBV vaccination was provided as appropriate. A total of 11,818 participants were interviewed, 11,572 samples collected, and 5,176 participants vaccinated from the 5 westernmost districts in Arunachal Pradesh. The overall hepatitis B surface antigen (HBsAg) prevalence was found to be 3.6% (n = 419). In total, 34.6% were hepatitis B e antigen positive (n = 145) and 25.5% had HBV DNA levels greater than 20,000 IU/mL (n = 107). Genotypic analysis showed that many patients were infected with HBV C/D recombinants. Certain tribes showed high seroprevalence, with rates of 9.8% and 6.3% in the Miji and Nishi tribes, respectively. The prevalence of HBsAg in individuals who reported medical injections was 3.5%, lower than the overall prevalence of HBV. CONCLUSIONS: Our unique, simplistic model of care was able to link a highly resource-limited population to screening, preventive vaccination, follow-up therapeutic care, and molecular epidemiology to define the migratory nature of the population and disease using an electronic platform. This model of care can be applied to other similar settings globally.

CD8+CD28- T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection.

During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28- T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28- T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28- T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28- T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28- T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28- T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28- T-cell killing. Both CD28+ and CD28- T cells in CHB expressed CXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28- T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28- T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28- T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28- T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

Distinctiveness in virological features and pathogenic potentials of subgenotypes D1, D2, D3 and D5 of Hepatitis B virus.

Distinct clinical features of HBV infection have been associated with different viral genotype/subgenotype. HBV Genotype-D comprised of 10 subgenotypes, D1-D10, whose clinical implications still remain elusive. We investigated for the first-time, the virologic characteristics and cytopathic effects of four non-recombinant D-subgenotypes, D1/D2/D3/D5. Expressions of viral/host genes were evaluated in Huh7 cells transfected with full-length, linear-monomers of HBV/D-subgenotypes or pGL3-Basic vector carrying subgenotype-specific HBx. Intracellular HBV-DNA and pregenomic-RNA levels were high in D1/D2 than D3/D5. Expressions of PreC-mRNA and HBx were highest for D2 and D1 respectively, whereas PreS2/S-transcript was significantly reduced in D5. Increased apoptotic cell death and marked upregulation in caspase-3/Bax/TNF-R1/FasR/TRAIL-R1/ROS/MCP-1/IP-10/MIP-1ß expression were noticed specifically in D2- and also in D3-transfected cells, while D5 resulted in over-expression of ER-stress-markers. D-subgenotype-transfected Huh7 cells were co-cultured with PBMC of healthy-donors or LX-2 cells and significant increase in pro-inflammatory cytokines in PBMC and fibrogenic-markers in LX-2 were noticed in presence of D2/D3. Further, Huh7 cells transfected with D1, in particular and also D5, displayed remarkable induction of EMT-markers and high proliferative/migratory abilities. Collectively, our results demonstrated that D2/D3 were more associated with hepatic apoptosis/inflammation/fibrosis and D1/D5 with increased risk of hepatocarcinogenesis and emphasize the need for determining HBV-subgenotype in clinical practice.

A rare HBV subgenotype D4 with unique genomic signatures identified in north-eastern India--an emerging clinical challenge?

BACKGROUND/AIMS: HBV has been classified into ten genotypes (A-J) and multiple subgenotypes, some of which strongly influence disease outcome and their distribution also correlate with human migration. HBV infection is highly prevalent in India and its diverse population provides an excellent opportunity to study the distinctiveness of HBV, its evolution and disease biology in variegated ethnic groups. The North-East India, having international frontiers on three sides, is one of the most ethnically and linguistically diverse region of the country. Given the paucity of information on molecular epidemiology of HBV in this region, the study aimed to carry out an in-depth genetic characterization of HBV prevailing in North-East state of Tripura. METHODS: From sera of chronically HBV infected patients biochemical/serological tests, HBV DNA quantification, PCR-amplification, sequencing of PreS/S or full-length HBV genomes were done. HBV genotype/subgenotype determination and sequence variability were assessed by MEGA5-software. The evolutionary divergence times of different HBV subgenotypes were estimated by DNAMLK/PHYLIP program while jpHMM method was used to detect any recombination event in HBV genomes. RESULTS: HBV genotypes D (89.5%), C (6.6%) and A (3.9%) were detected among chronic carriers. While all HBV/A and HBV/C isolates belonged to subgenotype-A1 and C1 respectively, five subgenotypes of HBV/D (D1-D5) were identified including the first detection of rare D4. These non-recombinant Indian D4 (IndD4) formed a distinct phylogenetic clade, had 2.7% nucleotide divergence and recent evolutionary radiation than other global D4. Ten unique amino acids and 9 novel nucleotide substitutions were identified as IndD4 signatures. All IndD4 carried T120 and R129 in ORF-S that may cause immune/vaccine/diagnostic escape and N128 in ORF-P, implicated as compensatory Lamivudine resistance mutation. CONCLUSIONS: IndD4 has potential to undermine vaccination programs or anti-viral therapy and its introduction to North-East India is believed to be linked with the settlement of ancient Tibeto-Burman migrants from East-Asia.

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

AIMS: The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored. METHODS: The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed. RESULTS: HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC. CONCLUSIONS: Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.

Genetic variants of TNFα, IL10, IL1ß, CTLA4 and TGFß1 modulate the indices of alcohol-induced liver injury in East Indian population.

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80 g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1ß -511CC genotype, TGFß1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFß1 loci among alcoholics. The risk genotype of IL1ß and TGFß1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1ß and TGFß1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.

"Normal" liver stiffness measure (LSM) values are higher in both lean and obese individuals: a population-based study from a developing country.

UNLABELLED: The liver stiffness measure (LSM) needs to be explored in ethnically and anthropometrically diverse healthy subjects (to derive an acceptable normal range) and also in patients with liver disease. In view of this objective, LSM was performed by transient elastography (TE) using FibroScan in 437 healthy subjects with normal alanine aminotransferase (ALT) levels, recruited from a free-living population of the Birbhum Population Project (BIRPOP; www.shds.in), a Health and Demographic Surveillance System (HDSS), and from 274 patients with liver disease attending the Hepatology Clinic of the School of Digestive and Liver Diseases (SDLD; Institute of Post Graduate Medical Education & Research [IPGME&R], Kolkata, India) including 188 with nonalcoholic fatty liver disease (NAFLD) and 86 with chronic hepatitis of viral and other etiologies. Liver biopsy was performed in 125 patients. The range of normal values for LSM, defined by 5th and 95th percentile values in healthy subjects, was 3.2 and 8.5 kPa, respectively. Healthy subjects with a lower body mass index (BMI; < <18.5 kg/m(2)) had a higher LSM compared with subjects who had a normal BMI; this LSM value was comparable to that of obese subjects (6.05 ± 1.78 versus 5.51 ± 1.59 and 6.60 ± 1.21, P = 0.016 and 0.349, respectively). Liver disease patients without histologic fibrosis had significantly higher LSM values compared with healthy subjects (7.52 ± 5.49 versus 5.63 ± 1.64, P < 0.001). Among the histologic variables, stage of fibrosis was the only predictor for LSM. LSM did not correlate with inflammatory activity and ALT in both NAFLD and chronic hepatitis groups. CONCLUSION: LSM varies between 3.2 and 8.5 kPa in healthy subjects of South Asian origin. Both lean and obese healthy subjects have higher LSM values compared with subjects with normal BMI. Liver stiffness begins to increase even before fibrosis appears in patients with liver disease.

Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice.

Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 µg/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-ß1, PDGF-Rß, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro(α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.

Course of disease and survival after onset of decompensation in hepatitis B virus-related cirrhosis.

BACKGROUND: Data regarding the outcome of hepatitis B virus (HBV)-related cirrhosis after the onset of decompensation is scanty. METHOD: From January 1998 to December 2008, a retrospective-prospective inception cohort study involving HBV-related decompensated cirrhotics was performed. Predictors of death and clinical events after the onset of decompensation were evaluated. Patients with co-infection with hepatitis C virus and/or human immunodeficiency virus, alcohol consumption to any degree and diabetes diagnosed before the detection of liver disease were excluded. RESULT AND ANALYSIS: Two hundred and fifty-three patients (231 males, 139 e-negative), including 102 untreated patients, were analysed. The mean (+/-SD) age was 43.0 (+/-12.0) years. The mean (+/-SD) follow-up period was 47 (+/-47) months. Decompensation was the first presentation of liver disease in 210 (83%) patients. Ascites (70%) and variceal bleed (28%) were predominant modes of decompensation. Forty-three (17%) patients died (22 vs 14% in untreated and treated cohort, respectively; P=0.002). Type 2 hepato-renal syndrome was the commonest cause of death (32%). Survival was independent of e-antigen status. In the total cohorts, predictors of death were occurrence of sepsis with systemic inflammatory response (SIRS), ascites as the initial mode of decompensation, absence of antiviral therapy and events of high-grade hepatic encephalopathy [hazards ratios (HR) of 4.4, 3.6, 2.2 and 1.7 respectively]. In the untreated cohort, initial decompensation with ascites and development of sepsis with SIRS were independent predictors of death (HR 8.5 and 2.3 respectively), while 5-year survival was higher in patients having initial decompensation with variceal bleed vs ascites (29 vs 16%, respectively, P=0.002). CONCLUSION: Decompensation with ascites and sepsis with SIRS predict reduced survival. Antiviral therapy beyond 6 months improves outcome.

Nonobese population in a developing country has a high prevalence of nonalcoholic fatty liver and significant liver disease.

UNLABELLED: There is a paucity of community-based epidemiological data on nonalcoholic fatty liver (NAFL) among nonaffluent populations in developing countries. Available studies are radiological and/or biochemical and lack histological assessment, limiting their strength. We conducted a prospective epidemiological study comprising a 1:3 subsample of all adult (>18 years) inhabitants of a rural administrative unit of West Bengal, India. Subjects positive for hepatitis B virus and/or hepatitis C virus infection and consuming any amount of alcohol were excluded. Diagnosis of NAFL was by dual radiological screening protocol consisting of ultrasonographic and computed tomographic examination of the liver. Transient elastographic examination and liver biopsy were performed in a subset to identify significant liver disease. The risk factors of having NAFL were analyzed. A total of 1,911 individuals were analyzed, 7% of whom were overweight and 11% of whom had abdominal obesity. The prevalence of NAFL, NAFL with elevated alanine aminotransferase, and cryptogenic cirrhosis was 8.7%, 2.3%, and 0.2%, respectively. Seventy-five percent of NAFL subjects had a body mass index (BMI) <25 kg/m(2), and 54% were neither overweight nor had abdominal obesity. The subjects with the highest risk of having NAFL were those with a BMI >25 kg/m(2) (odds ratio 4.3, 95% confidence interval 1.6-11.5). Abdominal obesity, dysglycemia (fasting plasma glucose >100 mg/dL or elevated homeostatic model assessment of insulin resistance), and higher income were the other risk factors. Even having a normal BMI (18.5-24.9 kg/m(2)) was associated with a 2-fold increased risk of NAFL versus those with a BMI <18.5 kg/m(2). CONCLUSION: There is a significant prevalence of NAFL and potentially significant liver disease, including cryptogenic cirrhosis, in this predominantly nonobese, nonaffluent population in a developing country. NAFL will be a major determinant of future liver disease burden in countries of the developing world.

Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine.

Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects.

Mitochondrial oxidative stress and permeability transition in isoniazid and rifampicin induced liver injury in mice.

BACKGROUND/AIMS: To evaluate the role of mitochondrial oxidative stress and permeability transition (MPT) in isoniazid (INH) and rifampicin (RMP) induced hepatotoxicity in mice. METHODS: Liver damage was induced by co-treatment of INH (50 mg/kg) and RMP (100 mg/kg). Pre-treatment with either methionine or phorone was done to modulate hepatic GSH level. Liver cell injury was assessed biochemically and histologically. Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. RESULTS: INH and RMP co-treatment caused steatosis and increased apoptosis of the hepatocytes, hepatic oxidative stress, particularly in the mitochondrial fraction with increased mitochondrial permeability transition (MPT). Mitochondrial oxidative stress as well as liver cell injury was increased by prior treatment with phorone. This was attenuated by pretreatment with methionine suggesting a glutathione (GSH) dependent phenomenon. CONCLUSIONS: Oxidative stress in the mitochondria and inappropriate MPT are important in the pathogenesis of apoptotic liver cell injury in INH-RMP hepatotoxicity. The phenomenon is GSH dependent and methionine supplementation might have a protective role.

Interaction between IL1B gene promoter polymorphisms in determining susceptibility to Helicobacter pylori associated duodenal ulcer.

It has been speculated that IL-1 genes play a crucial role in the genetic predisposition to duodenal ulcer upon H. pylori infection by modulating the host immune response. In the present study, 310 individuals from Eastern India were subjected to a case-control study to determine the IL1B and IL1RN risk genotypes to H. pylori mediated duodenal ulcer. An analysis of genotype frequency revealed a significantly higher frequency of IL1B -511TT (NT_022135.14:g.2302610C>T), OR=4.22 (95% CI=1.8-9.4) and -31CC (NT_022135.14:g.2302130C>T), OR=2.16 (95% CI 1.12-4.15) genotypes in H. pylori-infected individuals with duodenal ulcer compared to infected individuals with normal mucosa. Moreover, the T/C haplotype of IL1B -511 and IL1B -31 loci was present in a significantly higher frequency in H. pylori-infected duodenal ulcer patients than in infected controls (OR=2.47, CI=1.27-4.8). Quantitative analysis of the mucosal IL1B mRNA revealed that among H. pylori-infected individuals, carriers of the -31CC genotype had significantly lower IL1B transcript levels than carriers of the CT (P<0.001) and TT (P<0.001) genotypes, independently of disease status. An IL1B promoter activity assay showed that the promoter with -31T had a 10-fold increase in activity compared to the one with -31C. The IL1B promoter bearing the different combinations of both polymorphic loci showed an interaction between the -511 and -31 loci. Our results show that H. pylori-infected individuals with the -31CC genotype secrete less IL1B and are susceptible to duodenal ulcers. They also suggest that the allelic interaction between the -511 and -31 polymorphic sites determines the overall strength of the IL1B promoter.

Diagnosis and genotyping of Helicobacter pylori by polymerase chain reaction of bacterial DNA from gastric juice.

BACKGROUND: Efficient and accurate detection of Helicobacter pylori infection as well as identification of virulence-associated alleles are important for the treatment of gastroduodenal diseases caused by this gastric pathogen. The present study was performed to test the efficiency of gastric juice polymerase chain reaction (PCR) method for the rapid detection of H. pylori infection and to determine the bacterial genotypes without the need for culture, which is often not feasible especially in developing countries. METHODS: DNA was extracted from gastric juice samples collected from 45 subjects and was used to amplify urease B gene (ureB) for H. pylori. Results obtained from this method were further confirmed by rapid urease test (RUT), histology and culture. Genotypes of the infected strains predicted from gastric juice PCR were compared to the genotype data obtained from the isolated strains. RESULTS: Among 45 cases, 32 were positive by RUT, 37 by histological examination, 25 by gastric juice PCR method, while culture yielded positive results for 19 samples. Except for one case, all the 19 culture-positive strains gave the same genotype with the gastric juice PCR result. It was found that the gastric juice PCR is more efficient for detection of multiple-strain infection as compared to genotype data obtained from strains isolated as pooled culture. CONCLUSIONS: This moderately sensitive technique could be employed with good efficiency, particularly in cases where it is difficult to obtain biopsy. Moreover, with this method bacterial genotype could be obtained.

Nucleic acid sequence analysis of basal core promoter/precore/core region of hepatitis B virus isolated from chronic carriers of the virus from Kolkata, eastern India: low frequency of mutation in the precore region.

OBJECTIVE: The aim of the present study was to characterize the predominant hepatitis B virus (HBV) strains and their molecular variants present in the HBV isolates of the different genotypes found among the chronic carriers of the virus in our community. METHODS: Precore/core and core promoter regions of HBV DNA were amplified by polymerase chain reaction and then subjected to direct sequencing. Of the 64 hepatitis B surface antigen (HBsAg)-positive chronic HBV carriers investigated, 44 were HBeAg negative and 20 were HBeAg positive. RESULTS: In addition to genotype D, which was the predominant genotype, 12 genotype C (18.7%) and 6 genotype A (9.4%) were also detected. Presence of T at nt 1858 has often been related to the development of precore stop mutation at nt 1896, while that of C has been related to the development of 1762-1764 double mutation. In our study group, 39 of the 44 HBeAg-negative samples have T1858. The precore stop codon mutation was found in only 8 (18%) of the HBeAg-negative samples. More than half of the HBeAg-negative samples had wild-type sequence in the precore region. The core promoter region could be sequenced from 40 samples, and 1762-1764 double mutation was detected in 13 (32.5%) of them. No significant changes could be detected in the core amino acid sequence of these isolates. CONCLUSION: The pattern of core promoter and precore mutation of HBV isolates in the present study is atypical and not in accordance with reports from other parts of the world, where genotype D and genotype C with T at codon 1858 are common.

Implications of oxidative stress and hepatic cytokine (TNF-alpha and IL-6) response in the pathogenesis of hepatic collagenesis in chronic arsenic toxicity.

INTRODUCTION: Noncirrhotic portal fibrosis has been reported to occur in humans due to prolonged intake of arsenic contaminated water. Further, oxystress and hepatic fibrosis have been demonstrated by us in chronic arsenic induced hepatic damage in murine model. Cytokines like tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) are suspected to play a role in hepatic collagenesis. The present study has been carried out to find out whether increased oxystress and cytokine response are associated with increased accumulation of collagen in the liver due to prolonged arsenic exposure and these follow a dose-response relationship. METHODS: Male BALB/c mice were given orally 200 microl of water containing arsenic in a dose of 50, 100, and 150 mug/mouse/day for 6 days a week (experimental group) or arsenic-free water (<0.01 microg/l, control group) for 3, 6, 9 and 12 months. Hepatic glutathione (GSH), protein sulfhydryl (PSH), glutathione peroxidase (GPx), Catalase, lipid peroxidation (LPx), protein carbonyl (PC), interleukin (IL-6), tumor necrosis factor (TNF-alpha), arsenic and collagen content in the liver were estimated from sacrificed animals. RESULTS: Significant increase of lipid peroxidation and protein oxidation in the liver associated with depletion of hepatic thiols (GSH, PSH), and antioxidant enzymes (GPx, Catalase) occurred in mice due to prolonged arsenic exposure in a dose-dependent manner. Significant elevation of hepatic collagen occurred at 9 and 12 months in all the groups associated with significant elevation of TNF-alpha and IL-6. However, arsenic level in the liver increased progressively from 3 months onwards. There was a positive correlation between the hepatic arsenic level and collagen content (r = 0.8007), LPx (r = 0.779) and IL-6 (r = 0.7801). Further, there was a significant negative correlation between GSH and TNF-alpha (r = -0.5336)) and LPx (r = -0.644). CONCLUSION: Increasing dose and duration of arsenic exposure in mice cause progressive increase of oxystress and elevation of cytokines associated with increasing level of collagen in the liver.

Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens.

We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.

Arsenic in drinking water and skin lesions: dose-response data from West Bengal, India.

BACKGROUND: Over 6 million people live in areas of West Bengal, India, where groundwater sources are contaminated with naturally occurring arsenic. The key objective of this nested case-control study was to characterize the dose-response relation between low arsenic concentrations in drinking water and arsenic-induced skin keratoses and hyperpigmentation. METHODS: We selected cases (persons with arsenic-induced skin lesions) and age- and sex-matched controls from participants in a 1995-1996 cross-sectional survey in West Bengal. We used a detailed assessment of arsenic exposure that covered at least 20 years. Participants were reexamined between 1998 and 2000. Consensus agreement by four physicians reviewing the skin lesion photographs confirmed the diagnosis in 87% of cases clinically diagnosed in the field. RESULTS: The average peak arsenic concentration in drinking water was 325 microg/liter for cases and 180 microg/liter for controls. The average latency for skin lesions was 23 years from first exposure. We found strong dose-response gradients with both peak and average arsenic water concentrations. CONCLUSIONS: The lowest peak arsenic ingested by a confirmed case was 115 microg/liter. Confirmation of case diagnosis and intensive longitudinal exposure assessment provide the basis for a detailed dose-response evaluation of arsenic-caused skin lesions.

Helicobacter pylori, gastric juice, and arterial ammonia levels in patients with cirrhosis.

Helicobacter pylori urease activity is a potential source of ammonia in the stomach of patients with cirrhosis. However, the possible role of H. pylori in the pathogenesis of hepatic encephalopathy deserves further investigations. The current study evaluates the relationship among H. pylori infection, gastric juice ammonia concentrations, and arterial ammonia levels in patients with cirrhosis. Overall, 14 patients with cirrhosis with overt hepatic encephalopathy, 19 with subclinical hepatic encephalopathy, and 13 without encephalopathy were enrolled. All patients underwent upper endoscopy, and gastric biopsy specimens were taken for H. pylori assessment (rapid urease test, histology, and culture). A gastric juice sample and an arterial blood sample were obtained for ammonia level assessment. Patients with overt encephalopathy had both higher arterial ammonia levels and a more severe hepatic impairment than the remaining patients, whereas gastric juice ammonia concentrations did not differ among the three groups. H. pylori prevalence was similar among groups. Patients with H. pylori infection had significantly higher gastric juice ammonia concentrations than those without infection (2.3 +/- 1.3 vs. 0.9 +/- 0.6 mmol/L, respectively; p = 0.003); however, no difference in arterial ammonia levels emerged between the two groups (37.7 +/- 18.6 vs. 37.6 +/- 18.8 micromol/L, respectively). No significant correlation was found between gastric juice ammonia concentrations and arterial ammonia levels. The data suggest that liver impairment remains crucial in ammonia disposal in patients with cirrhosis, whereas H. pylori infection does not seem to play a major role in the pathogenesis of hyperammonemia in these patients.