Molecular medicine techniques in the detection of viral hepatitis

Viral hepatitis is a major concern in the public health scenario which has been the root cause of serious adverse events affecting the human population. Molecular diagnostics has paved the way for an incredible role in the advancement of the prognosis and the diagnosis of viral hepatitis. Significant improvements and modifications in the techniques have led to a breakthrough in identifying the various reasons underlying the severity of the various hepatitis viruses causing acute and chronic infections besides cancer. The molecular biological analysis of the infectious agents involves detailed investigation of their genomes and their genomic products. Polymorphism studies have unleashed many important associations of the diseases with the various genetic variants. Besides these, recombinant DNA technology, modified sequencing technologies, rational drug designing have revolutionized the clinical sphere with their varied applications along with an immense impact on human health. Through the development of novel genetic methodologies, including a better perception of pathogen biology, pathogenic mechanisms, recent advances in vaccine development, scheming new therapeutic drugs and optimization of diagnostic tools, viral hepatitis caused by viral infections are now better controlled. In this brief review we discuss the developments in the various diagnostic tools in the detection of the viral infections inflicting the liver in the preceding years along with some recent upcoming techniques which may revolutionize the treatment and diagnosis of viral hepatitis caused by various viral infections

Detection of Nucleoside/Nucleotide Drug Resistant Mutants in Liver Cancer Cases: An Experience from India

Abstract Use of nucleoside and nucleotide analogues to treat patients infected with hepatitis B virus (HBV) has been found to be associated with mutations in the polymerase gene of the virus. The current study was carried out in HBV-related hepatocellular carcinoma (HCC) cases to trace the presence of drug-related mutants. A total of 75 HBV-related HCC cases were included for the study as per Bruix et al., 2001 EASL guidelines. HBV viral DNA was isolated by the previously standardized manual phenol-chloroform methods. The 3.2 kb genome of HBV was amplified by six sets of overlapping primers. The amplicons were sequenced commercially [Macrogen, South Korea (ABI PRISM)]. Sequences for the polymerase gene were analyzed using commercial bioinformatics software (http://www.hepseq.org/Public/Tool/annotator_tool.php). The different drug-resistant mutations detected were confirmed twice, ahead of reporting. Four drug-resistant mutations were detected in total: L80I (lamivudine), N236T (adefovir), I169T (entecavir) and A181V (lamivudine/adefovir). Interestingly, all four of the drug-resistant mutants were found in genotype D of HBV. The low number (only four) of drug-resistant mutations detected in this study population can be attributed to the fact that most of the cases were not treated and presented late. This study's findings confirm the presence of previously reported drug-resistant mutations in the HBV genome infecting Indian patients; however, its associations with late stage disease and with the virus genotype D, in particular, need to be further studied in a larger population

Immunological and molecular epidemiological characteristics of acute and fulminant viral hepatitis A

<p>Abstract</p> <p>Background</p> <p>Hepatitis A virus is an infection of liver; it is hyperendemic in vast areas of the world including India. In most cases it causes an acute self limited illness but rarely fulminant. There is growing concern about change in pattern from asymptomatic childhood infection to an increased incidence of symptomatic disease in the adult population.</p> <p>Objective</p> <p>In-depth analysis of immunological, viral quantification and genotype of acute and fulminant hepatitis A virus.</p> <p>Methods</p> <p>Serum samples obtained from 1009 cases of suspected acute viral hepatitis was employed for different biochemical and serological examination. RNA was extracted from blood serum, reverse transcribed into cDNA and amplified using nested PCR for viral quantification, sequencing and genotyping. Immunological cell count from freshly collected whole blood was carried out by fluorescence activated cell sorter.</p> <p>Results</p> <p>Fulminant hepatitis A was mostly detected with other hepatic viruses. CD8⁺T cells count increases in fulminant hepatitis to a significantly high level (P = 0.005) compared to normal healthy control. The immunological helper/suppressor (CD4⁺/CD8⁺) ratio of fulminant hepatitis was significantly lower compared to acute cases. The serologically positive patients were confirmed by RT-PCR and total of 72 (69.2%) were quantified and sequenced. The average quantitative viral load of fulminant cases was significantly higher (<it>P </it>< 0.05). There was similar genotypic distribution in both acute and fulminant category, with predominance of genotype IIIA (70%) compared to IA (30%).</p> <p>Conclusions</p> <p>Immunological factors in combination with viral load defines the severity of the fulminant hepatitis A. Phylogenetic analysis of acute and fulminant hepatitis A confirmed genotypes IIIA as predominant against IA with no preference of disease severity.</p

Tumour necrosis factor-a and soluble Fas ligand as biomarkers in non-acetaminophen-induced acute liver failure

Objectives: Cytokines as prognostic markers in acute liver failure (ALF) have not been evaluated in the Indian subcontinent with hepatitis E as the commonest aetiological agent. We investigated the clinical significance of proinflammatory/apoptotic cytokines soluble Fas ligand (sFasL) and tumour necrosis factor (TNF)-&#945; in ALF of specific aetiology. Methods: A total of 82 cases, 37 ALF and 45 acute hepatitis (AH), and 60 healthy controls were recruited. Serum levels of sFasL and TNF-&#945; were determined at admission and death/recovery. Results: Mean sFasL and TNF-&#945; serum levels at admission were significantly higher (p &lt; 0.001) in patients with ALF than AH, but no marked difference was observed between ALF-E (expired, n=23) and ALF-S (survivors, n=14), although the former had comparatively higher levels. ALF-E had higher than baseline TNF-&#945; and sFasL concentrations at death, while in the ALF-S group the samples obtained from the patients as soon as they came out of encephalopathy, showed either lower or similar TNF-&#945; and sFasL levels as found at admission. Conclusion: The high levels of sFasL and TNF-&#945; are associated with ALF. Following the trend of these cytokines can be useful in predicting death and timely referral to a transplant centre

Hepatitis B virus genotypes in acute and fulminant hepatitis patients from North India using two different molecular genotyping approaches

Data from India on hepatitis B virus (HBV) genotype related differences in clinical progression and outcome of acute and fulminant hepatitis B are limited. Sera from patients with acute hepatitis B (AHB) (n = 80), fulminant hepatitis B (FHB) (n = 40) and asymptomatic HBsAg carriers (ASC) (n = 40) were tested for HBV genotype using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and type-specific primers-based PCR (TSP-PCR). The genotype distribution for 160 patients with HBV related hepatitis/carriers were as follows: A, 3/80 (3.7%) in AHB, 2/40 (5%) in FHB and 7/40 (17.5%) in ASC; D, 77/80 (96.2%) in AHB, 38/40 (95%) in FHB and 33/40 (82.5%) in ASC. C, 0; B, 0; E, 0; F, 0 (p &#60; 0.01, genotype D versus A). Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (p &#62; 0.05). HBV genotypes A and D were found to be prevalent in patients with HBV related acute and fulminant hepatitis from New Delhi, India. Genotype D was the dominant genotype prevalent in all patient categories while genotype A was solely responsible for AHB leading to chronic hepatitis B in 3.7% of the cases from this region