Bhupesh Singla

Small intestinal bacterial overgrowth and toll-like receptor signaling in patients with non-alcoholic fatty liver disease

The pathogenesis of non‐alcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll‐like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD.

Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre‐existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi‐nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment‐naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed. J. Med. Virol. 85:1155–1162, 2013.

Angiogenic and anti-angiogenic factor gene transcript level quantitation by quantitative real time PCR in patients with hepatocellular carcinoma

Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. The aim of this study was to quantify the expression of angiogenic (VEGF, HIF-1α, Angiopiotein-2) and anti-angiogenic (endostatin, angiostatin and Thrombospondin-1) factors and to discern their clinical relevance. A total 90 patients (67 HCC, 9 cirrhosis and 14 chronic hepatitis) were enrolled in the study. Tissue transcript levels of angiogenic (VEGF, HIF-1α, Ang-2) and anti-angiogenic (endostatin, angiostatin and TSP-1) factors were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR) in the tissue samples. The tissue transcript levels of VEGF, HIF-1α and endostatin were found to be significantly higher in HCC in comparison to cirrhosis and chronic hepatitis. Although Ang-2, angiostatin and TSP-1 tissue transcript levels were higher in HCC group than the others groups but the difference was not statistically significant. In univariate analysis both VEGF and HIF-1α were found to be associated with poor survival of HCC patients. Multivariate analysis by the cox proportional hazard model revealed only VEGF as an independent factor predicting poor survival of the HCC patients. Angiogenic and anti-angiogenic factors are all highly expressed in HCC patients. Upregulation of tissue anti-angiogenic factors indicates the urgency for the alternative of anti-angiogenic therapies.

Genetic polymorphism in CD14 gene, a co-receptor of TLR4 associated with non-alcoholic fatty liver disease.

AIM To evaluate the pathogenic role of toll-like receptor (TLR) gene polymorphisms in patients with non-alcoholic fatty liver disease (NAFLD). METHODS Two hundred and fifty subjects (NAFLD = 200, healthy volunteers = 50) underwent polymerase chain reaction and restriction fragment length polymorphism to assess one polymorphism in the toll-like receptor 2 (TLR2) gene (A753G), two polymorphisms in the TLR4 gene (TLR4 Asp299Gly and Thr399Ile allele), and two polymorphisms in the cluster of differentiation 14 (CD14) (C-159T and C-550T) gene, a co-receptor of TLR4. Association of TLR gene polymorphisms with NAFLD and its severity was evaluated by genetic models of association. RESULTS On both multiplicative and recessive models of gene polymorphism association, there was significant association of CD14 C (-159) T polymorphism with NAFLD; patients with TT genotype had a 2.6 fold increased risk of developing NAFLD in comparison to CC genotype. There was no association of TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, and CD14 C (-550) T polymorphisms with NAFLD. None of the TLR gene polymorphisms had an association with histological severity of NAFLD. CONCLUSION Patients with CD14 C (-159) T gene polymorphism, a co-receptor of TLR4, have an increased risk of NAFLD development.

Response to potent anti-HBV agents in chronic hepatitis B and combined effect of HBV reverse transcriptase mutations.

BACKGROUND AND AIM Response to nucleos(t)ide analogue therapy against HBV infection depends on a number of factors. One of them is appearance of drug resistance mutations. The present study aimed to investigate the efficacy of ETV and TDF as anti-HBV agents and to analyze the role of HBV-RT mutations in reducing the efficacy of mentioned drugs. MATERIAL AND METHODS Sixty nine treatment naïve CHB patients (mean age 33.8 ± 11.9 years) were enrolled and treated with ETV or TDF for one year. Complete virological response (CVR) was defined as undetectable serum HBV DNA after 12 months of therapy. Amino acid and nucleotide sequence analyses of HBV-RT region were performed using Geno2pheno HBV drug resistance tool. The 3D model of HBV-RT protein was built by I-TASSER server and RMSD was calculated between wild type and mutated HBV-RT protein. RESULTS After 12 months of treatment, four CHB patients did not achieve CVR and all of them were with HBV genotype D. HBeAg seroconversion was achieved in 56% HBeAg positive patients after 12 months of antiviral therapy. The HBV-RT amino acid sequences from these four patients were used for in-silico analysis. It was found that the presence of many mutations in HBV-RT region of HBV isolated from these patients led to a high degree of variation in configuration of atoms of HBV-RT protein and also caused displacement of active site of this protein. CONCLUSION The efficacy of antiviral drugs in inhibiting HBV replication may be reduced by combined effect of many HBV-RT mutations; however, an in vitro study is needed to validate the findings.

P37: Quantification of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients

PURPOSE OF THE STUDY: The monitoring of cccDNA levels can provide a direct indication of HBV activity in the liver of HBV infected patients. The aim of this study was to quantify the cccDNA levels in sera and intrahepatic levels of HBV DNA and cccDNA in liver biopsies of treatment na€ıve patients with chronic hepatitis B. METHOD: Eighty one chronic HBV treatment na€ıve patients were enrolled from January 2009 to June 2011. The levels of intrahepatic HBV DNA and cccDNA were quantified using real time PCR method. SUMMARY OF RESULTS: The mean age of recruited patients was 34 11.5 years. A total of 54 patients (66.7%) were HBeAg negative. Liver biopsy was done in 23 patients (21 HBeAg negative and 2 HBeAg positive). The levels of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA were 0.31 copies/cell (range 0.14–0.49 copies/cell) and 0.20 copies/cell (range 0.01–1.63 copies/cell) in HBeAg positive and HBeAg negative cases, respectively. The rate of serum HBV cccDNA detection was 85.2% and 48.1% in HBeAg positive and negative patients, respectively. The median level of serum HBV cccDNA was 46,000 copies/ mL in HBeAg positive cases, while it was 26,350 copies/ mL in HBeAg negative disease. The levels of intrahepatic HBV total DNA had a positive correlation with intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was observed between serum cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). CONCLUSION: It was concluded that serum HBV DNA and serum cccDNA levels were significantly higher in HBeAg positive patients than HBeAg negative patients.