Post cholecystectomy hemobilia: transcatheter embolization of pseudoaneurysms with homemade steel coils

Two patients presented with hemobilia, one and two months following cholecystectomy. Angiography demonstrated pseudoaneurysms arising form the gastroduodenal and right hepatic arteries. Percutaneous transcatheter embolization of the pseudoaneurysms was successfully performed in both patients using homemade steel coils

Minimal hepatic encephalopathy: consensus statement of a working party of the Indian National Association for study of the liver

Hepatic encephalopathy (HE) is a major complication that develops in some form and at some stage in a majority of patients with liver cirrhosis. Overt HE occurs in approximately 30-45% of cirrhotic patients. Minimal HE (MHE), the mildest form of HE, is characterized by subtle motor and cognitive deficits and impairs health-related quality of life. The Indian National Association for Study of the Liver (INASL) set up a Working Party on MHE in 2008 with a mandate to develop consensus guidelines on various aspects of MHE relevant to clinical practice. Questions related to the definition of MHE, its prevalence, diagnosis, clinical characteristics, pathogenesis, natural history and treatment were addressed by the members of the Working Party

Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control

A method for quantitation of hepatitis C virus (HCV) RNA was developed based on competitive reverse transcription polymerase chain reaction (RT-PCR) using in vitro transcribed mutated thio-RNA as a competitor template. The thio-RNA is more resistant to RNAse and is stable over a year. This assay was compared with the commercially available Roche Amplicor HCV Monitor assay V 2.0 and real time PCR using SYBR green 1 dye method. A total of 18 pre-therapy serum samples from chronic hepatitis C cases were tested in parallel by the three assays. All samples could be quantitated using the in-house competitive RT-PCR and real time PCR and there was a significant correlation in the virus titer (P<0.05). However, 8 (44%) samples could not be quantified by Amplicor HCV Monitor assay, which has a lower detection range (102 to 105.5 copies/ml). The in-house method of competitive RT-PCR showed a detection range of 103 to 1010 copies/ml. In the patients the mean viral titer was found to be (9.66± 9.3)× 106 copies/ml. Ten (55%) of the samples, assessed by the Amplicor HCV Monitor assay showed a mean viral titre of (1.13± 0.75)× 106 copies/ml, which was lower than the other two tests. The competitive PCR method and real time PCR could amplify all prevalent genotypes. This in-house quantitative competitive RT-PCR method is simple, cheap, reproducible and useful for estimation of HCV RNA load

Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection

Background & objectives : Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated. Methods : We examined the in vitro expression of HBsAg, ratio of cure and ′e′ antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection. Results : Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and ′e′ antigen secretion. Interpretation & conclusions : The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication

Hepatitis D virus (HDV) infection in severe forms. Introduction of liver diseases in north India

Background: Preliminary reports indicate that hepatitis D virus (HDV) infection exists in India. However, its prevalence in patients with different types of liver diseases has not been studied in detail. The aim of this study was to evaluate the status of HDV infection in severe types of liver disease in India. Methods: Using commercial kits for various hepatitis viral markers, the present study was undertaken to determine the serological status of hepatitis B virus (HBV) and hepatitis D virus (HDV) markers in 208 patients with severe liver diseases. This total included 110 cases with fulminant hepatic failure (FHF), 65 cases with subacute hepatic failure (SHF) and 33 cases with chronic active hepatitis (CAH). Results: The hepatitis B surface antigen (HBsAg) carrier population, indicated by the presence of HBsAg without IgM anti-HBc (hepatitis B core) in serum, was recorded in 23.6%, 24.6% and 60.6% cases of FHF, SHF and CAH groups, respectively. HBV infection, as indicated by serum positivity of IgM anti-HBc in the FHF and SHF groups and HBsAg and/or IgM anti-HBc in the CAH group, was detected in 19.1%, 23.1% and 69.7% of cases from these three groups, respectively. IgM anti-HDV, demonstrating active/recent HDV infection, was found in 8.1% cases of FHF and 9.2% cases of SHF patients. HDV as a superinfection in HBsAg carriers was noted in 4.5% and 4.6% cases, respectively of FHF and SHF groups. Similarly, HDV-HBV coinfection, diagnosed by simultaneous presence of IgM anti-HBc and IgM anti-HDV in the FHF and SHF groups, was recorded in 3.6% and 4.6% of cases from these two groups, respectively. In the CAH group, HDV infection was observed in 9.2% cases. Conclusion: HDV infection, recorded in less than 10% of patients with different liver diseases in India, seems to be an unimportant factor in inducing severe liver diseases in this country

Efficacy of L-ornithine L-aspartate in acute liver failure: a double-blind, randomized, placebo-controlled study

Background & Aims: In acute liver failure (ALF), high blood ammonia levels have been documented that correlate with mortality and complications. L-ornithine L-aspartate (LOLA) reduces ammonia levels by increasing hepatic ammonia disposal and its peripheral metabolism. Present study evaluated efficacy and ammonia lowering effect of LOLA in ALF. Methods: This study was placebo-controlled and blinded. We randomized 201 patients with ALF between January 2005 and October 2007 to either placebo or LOLA infusions (30 g daily) for 3 days. Arterial ammonia was measured at baseline and daily for 6 days. The primary end point was improvement in survival. The study followed CONSORT guidelines and was registered at the ClinicalTrials.gov (Identifier: NCT00470314). Results: There was no reduction in mortality with LOLA treatment (mortality: 33.3% in placebo and 42.4% in LOLA; relative risk of death 1.27; 95% CI: 0.88-1.85; P = .204). By multivariate analysis, ammonia levels were an independent predictor of survival. There was significant decrease in ammonia levels in both groups with time (P < .001), but the levels of ammonia between the randomized groups at any time point, either during the 72 hours of LOLA infusion or during the follow-up were similar (P = .492). There was no difference between the 2 groups in the improvement in encephalopathy grade (P = .418), consciousness recovery time (P = .347), survival time (P = .612), or complications like seizures (P = .058) and renal failure (P = .615). The fetal outcome was also similar (P = .172). No adverse drug effect was noted. Conclusions: LOLA infusion did not lower the ammonia or improved survival in ALF

Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter

Background and Aims: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. Methods: Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. Results: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.1-2, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.3-4). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. Conclusions: These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative

Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA)

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-β and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%)

Role of surface promoter mutations in hepatitis B surface antigen production and secretion in occult hepatitis B virus infection

The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection

Protracted viremia during acute sporadic hepatitis E virus infection

Background/Aims: Hepatitis E virus (HEV) is associated with epidemic and sporadic hepatitis in developing countries. The disease is largely self-limited with no long-term sequelae. The source of HEV for maintenance of the disease in an endemic area is unknown. This study investigated the occurrence and duration of viremia in patients with acute sporadic HEV infection. Methods: In 26 of 37 patients with sporadic acute non-A, non-B viral hepatitis, HEV infection was diagnosed based on positivity for immunoglobulin M anti-HEV and/or presence of viremia as shown by reverse-transcription polymerase chain reaction. In 4 patients, fecal samples were analyzed for presence of virus using polymerase chain reaction. Multiple samples were studied at varying times in 20 patients. Results: Viremia was detected in 19 of 26 patients. Two patients had viremia in the absence of immunoglobulin M anti-HEV. Four patients had protracted viremia of 45-112 days' duration. One patient showed fecal virus shedding up to the 52nd day of illness. Conclusions: Protracted viremia and prolonged fecal shedding of HEV were shown in a small group of patients. These patients may serve as temporary virus carriers responsible for continuous contamination of the sewage water