Indian J Gastroenterol (November 2013) 32(Suppl 1):A133–A141 DOI 10.1007/s12664-013-0416-0

ABSTRACTS

Indian National Association for Study of the Liver

Received: 21 September 2013 / Accepted: 2 October 2013 / Published online: 6 November 2013 # Indian Society of Gastroenterology 2013

Viral Hepatitis INASL-1 Prevalence of hepatitis B and hepatitis C in diabetes mellitus

prevalence of anti-HBs. There was no significant difference in HBsAg seropositivity as compared to nondiabetic population. The rate of anti-HCV was higher in DM patients than nondiabetic population, however no significant difference was found. INASL-2

Rajiv Baijal, Praveen Kumar, Nimish Shah, Sandeep Kulkarni, Soham Doshi, Deepak Gupta, Deepak Amarapurkar Jagjivan Ram Railway Hospital, Maratha Mandir Marg, Behind Maratha Mandir Cinema, Mumbai Central, Mumbai 400 008, India Background: There is a paucity of studies on prevalence of hepatitis B and C in diabetes mellitus (DM) patients in India. The aim of our study was to determine the seroprevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in DM patients as compared to nondiabetic population. Methods: The study population included Western Railway employees and their families. All subjects were tested for blood sugar level, hepatitis B surface antigen (HBsAg), total anti-HBc, anti-HBs titre and anti-HCV. Results: We tested 1,474 adult subjects which included 364 diabetic patients. There was no significant difference (p-value 0.83) in HBsAg seropositivity in DM population (1.37 %, 5/364) and in control population (1.53 %, 17/1,110). AntiHCV positivity was 0.55 % (2/364) in diabetic patients and 0.09 % (1/1,110) in control patients with odds ratio of 6.12 and p-value 0.091 which was non-significant. Anti-HBs titre positivity was 16.20 % (59/364) in diabetic patients and 26.49 % (294/1,110) in control group with significant p -value (0.001). Anti-HBc total positivity was 23.9 % (87/364) in diabetic patients and 8.82 % (98/1,110) in control group with odds ratio of 3.24 and significant p-value (0.001). Conclusion: In DM patients, there was significant exposure to hepatitis B (higher prevalence of anti-HBc) with low

A novel loop mediated isothermal amplification assay for detection of HBV DNA: A pilot Study Nayana Joshi, V Lakshmi, Lavanya Vanjari, Neeraja, Priyanka, B Sukanya, Chintan Kansagra, Sandip Shah, Nikhil Shirole, Kunal Vyawahare, Ajit Kumar Department of Medical Gastroenterology, Nizam’s Institute of Medical Sciences, Punjagutta, Hyderabad 500 082, India Background: Real time PCR is gold standard for detection of HBV DNA, but is expensive, requires sophisticated equipment. Developing assays like loop mediated isothermal amplification (LAMP) which are cost effective, single tube assay with no sophisticated equipment are highly desirable. LAMP uses 6 different primers which specifically detect 8 different regions. Aim: 1. To develop LAMP for detection of HBV DNA. 2. To compare the results of LAMP assay with real time PCR. Materials and Methods: Plasma samples of chronic HBV carriers with known viral load were used for LAMP assay. Precore and e antigen region of HBV DNA was used for designing a set of 6 LAMP primers (2 each of outer, inner and loop). Primers were designed using primer explorer V4 software Eikengenome http://primererxplorer.jp/e. LAMP assay was performed at total 25 μL reaction volume using Bst polymerase. The amplification was observed using 1 μL of SYBR Green dye. Positive amplification is indicated by green fluorescence.

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Results: Eighteen samples (13 HBV DNA positive samples and 6 with DNA load less than 3.6 IU/mL) were tested for LAMP. All DNA positive samples were tested positive with LAMP. Amongst 6 negative samples, 2 were LAMP positive and 4 were LAMP negative. Conclusion: LAMP is a novel nucleic acid amplification method which is simple, rapid and cost effective. Detection rate of LAMP is comparable or higher than real time PCR, probably due to its lower limit of detection. This method may prove to be a useful tool in blood banks, resource poor settings and field applications. INASL-3 HCV treatment outcome in HIV coinfected patient in Western India: An observational study Atul K Patel, Ketan K Patel, Drupad Patel Infectious Diseases Clinic, Vedanta Institute of Medical Sciences, Navrangpura, Ahmedabad 380 009, India Background: Hepatitis C coinfection in HIV infected patients varies from 1.3 % to 9.2 % in different states in India. In HIV infected patients, HCV coinfection increases progression to AIDS, impairs immune reconstitution, and increases liverrelated deaths. Effective HCV treatment in HIV coinfected patient reduces liver related death and new AIDS defining events. Methods: It is a retrospective study of HIV-HCV coinfected patients treated for HCV infection with pegylated interferon+ ribavirin from year 2002 to 2011 at Infectious Diseases Clinic, Ahmedabad. All patients received ART for HIV infection and had stable regimen with undetectable HIV on viral load assay before starting HCV treatment. Data were analyzed using simple statistical methods. Results: Total 22 patients were included in the study. Twelve (54.54 %) patients received Peg-IFN alpha 2a (180 mcg) and 10 (45.45 %) Peg-IFN alpha 2b (weight based) along with ribavarin 800-1000 mg/day). Over all 36.36 % patients, 3 patients with genotyope 1 and 5 with genotype 3 achieved SVR. Four G3 patients had relapse after ETR. Various complications observed were anemia (90.90 %), leucopenia (81.81 %), thrombocytopenia (86.36 %), fever (77.27 %), weight loss (86.36 %), psychiatric disturbances (9.09 %), seizure (9.09 %) and death (13.63 %). Causes of death were brain tumor, complete heart block and colitis with sepsis in one patient each. RVR and EVR were seen in 45.5 % and 54.5 % patients respectively. Over all 36.36 % patients, 3 patients with genotyope 1 and 5 with genotype 3 achieved SVR. Four G3 patients had relapse after ETR. Various complications observed were anemia (90.90 %), leucopenia (81.81 %), thrombocytopenia (86.36 %), fever (77.27 %), weight loss (86.36 %), psychiatric disturbances (9.09 %), seizure (9.09 %) and death (13.63 %).

Indian J Gastroenterol (November 2013) 32(Suppl 1):A133–A141

Conclusions: HCV treatment in HIV coinfected patients is challenging due to variety of reasons. SVR was achieved in nearly 1/3rd patients. Management of complications and various drug-drug interactions requires great attention and skill as well as co-operation from patient. INASL-4 Treatment of chronic hepatitis C in patients with end stage renal disease Ramit Mahajan, Jeyamani Ramachandran, P L Alagammai, G Basu, V Tamilarasi Departments of Hepatology and Nephrology, Christian Medical College, Vellore 632 004, India Aim: To analyze treatment outcomes in patients with hepatitis C and end stage renal disease. Methods: Out of the 429 hepatitis C patients managed from June 2010-June 2012, 47 had chronic kidney disease. Treatment details and outcomes at various time points such as like rapid viral response (RVR), early viral response (EVR) end of treatment response (ETR) and sustained viral response (SVR) were assessed. Results: Eighteen of 45 patients with CKD (40 %) consented for treatment. Five patients dropped out due to various reasons. In the remaining 13 (27 %) patients, there were 9 men and 4 women; median age was 35 years (21-60). Except 2 patients (stage 3), the rest were in end stage renal disease (ESRD). Four patients had genotype 1, 8 had genotype 3 and indeterminate in one. Eight patients received conventional and five received pegylated interferon. Four patients took ribavarin in low doses. RVR (done in 5/13) was noted in 4 patients. EVR was achieved in 12/13 patients (92 %). ETR (data in 9 patients) was achieved in 7 (77 %) patients, negative in 2 patients and is awaited in 2 patients. Two patients were lost to follow up after EVR. Positive SVR was seen in 6 out of the 7 (85 %) with ETR, irrespective of the type of interferon used. Only one of them had genotype 1. Conclusions: Though only 27 % of patients with HCV and ESRD received antiviral therapy, excellent end of treatment and sustained viral response was seen in up to 80 %. INASL-5 Molecular epidemiology of hepatitis C virus from India Rahamathulla Syed, Raju Nagarapu, A Anusha, Aejaz Habeeb Mohammed, V V Ramachandra Rao, Madhavi Chandra, Aleem Ahmed Khan,Vishnupriya Satti, Khaja Mohammed Nanne Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500 058, and Department of Genetics, Osmania University, Tarnaka, Hyderabad 500 007, India

Indian J Gastroenterol (November 2013) 32(Suppl 1):A133–A141

Background: Hepatitis C virus (HCV) infection is becoming a major menace and is evolving as a public health problem globally. Studies on the prevalence of HCV in different countries suggest that more than 300 million people worldwide are infected, out of which at least 20 million people reside in India. Aims: Study molecular epidemiology of HCV in Andhra Pradesh, India. Results: The population size in the present investigation is 3, 168 which include 194 (6.12 %) individuals positive for HCV infection as detected by RT-PCR method, but the third generation ELISA detected only 126 (3.97 %) positive individuals. Among the 586 patients who attended the gastroenterology outpatient unit, 5 (0.85 %), 6 (1.02 %) individuals were positive for HCV by ELISA and RT-PCR methods respectively. Out of 1,698 voluntary blood donors screened, 23 (1.35 %) were positive in ELISA, while 28 (1.64 %) were positive by RT-PCR method. In the risk group category total number of patients screened were 877, out of which 98 (11.17 %) and 160 (18.24 %) were positive by ELISA and RT-PCR methods respectively. Genotypic distribution of 194 HCV patients in various categories of population. Among 6 patients attended gastroenterology camps, genotype 1a was found in 1 patient, genotype 1b in 1, genotype 3a in 1, genotype 3b in 1 patient, mixed genotypes in 2 patients. Among 28 blood donors genotype 1a was found in 2, genotype 1b in 13, genotype 3a in 2, genotype 3b in 10, and one new genotype 5a was reported. Genotyping in risk groups revealed that genotype 1b with 45.36 % is predominant followed by genotype 3b with 24.74 %. Conclusion: The data in various population groups with respect to HCV infection are likely to provide new insight in the causation of liver disease in India. The differential prevalence that has been observed in our study appears to be linked to the geographic areas of origin. This is the first report of Genotype 5a from India. INASL-6 Development of in vitro model for hepatitis C virus infection in human hepatocyte progenitors Aleem Ahmed Khan, Raju Nagarapu, Sandeep Kumar Viswakarma, Rahamathulla Syed, Avinash Bardia, Anusha Aggi, Ganga Bhavani, Aejaz Habeeb Mohammed Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500 058, India Background: Hepatitis C virus (HCV) is a major health concern causing chronic liver disease in humans. Understanding the pathogenesis of HCV infection in vivo presents several hurdles for the development of more appropriate treatment modalities.

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Aim: Development of in vitro culture model of HCV infection in primary hepatocytes may provide a significant tool to study the mechanism of pathogenesis of HCV in acute and chronic liver diseases. Methods: In present study EpCAM positive human hepatic progenitor cells were cultured in vitro and transfected with different genotypes of HCV serum and RNA samples. In vitro proliferation and survival of cells was determined using MTT assay, changes in cell morphology was identified using phase contrast microscopy. The transfection efficiency of HCV infection was determined by identification of viral RNA amplification using RT-PCR. Results: Transfected hepatocytes expressed HCV core proteins and HCV negative-strand RNA. For 2 months, transfected or infected cultures released HCV into the medium at high levels. Medium from transfected cells was able to infect naïve cultures in a Transwell system, Hepatic progenitors transfected with HCV serum of genotype 3a exhibited the highest viral titer, 4.2 × 10^6 HCV RNA copies/mL, at 18 days in culture. In contrast, HCV could not be detected in cultures inoculated with heat-inactivated serum. Conclusions: The study provides a better understanding about the pathogenesis, viral replication, and in vitro susceptibility of HCV. Characterization of HCV/serum infected in vitro model will give a clear image for the study of HCV infection. It also demonstrates the response of hepatocytes with different genotypes and HCV infected serum samples.

Nonalcoholic Fatty Liver Disease INASL-7 Lactose intolerance in nonalcoholic steatohepatitis patients from North India S V Rana, A Duseja, S Sharma, A Malik, Y K Chawla Departments of Super Specialty of Gastroenterology, and Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India Background: NASH is defined as accumulation of lipid in form of triacylglycerols in individuals who do not consume significant amounts of alcohol and in whom other known causes of steatosis, such as certain drugs and toxins, have been excluded. It has been reported in literature that NASH patients have higher prevalence of small intestinal bacterial overgrowth. Therefore, it is hypothesized that SIBO may lead to lactose intolerance in NASH patients. Hence, this study was planned to find incidence of lactose intolerance in NASH patients. Methods: Thirty patients of NASH with age range 22-65 years and 35 age and sex matched apparently healthy controls

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(age range 24-67 years) were enrolled. Noninvasive lactose hydrogen breath test was done in all subjects after overnight fast. For lactose hydrogen breath test, 25 g lactose dissolved in 250 mL water was given orally to subjects after taking fasting breath H2 and CH4 concentrations. End expiratory breath was collected every 30 minutes up to 4 hrs. Concentration of hydrogen and CH4 was measured by using SC microlyser from Quintron, USA. Rise >20 ppm over base line value in H2 concentration and/or CH4 in two consecutive readings was considered as lactose intolerance. Results: Out of 30 NASH, 10 (33.3 %) were females and 20 (66.6 %) were males while out of 35 healthy controls, 13 (37.2 %) were females and 22 (62.8 %) males. Twenty-one out of 30 (70 %) NASH were lactose intolerant, while in controls, 14 out of 35 (40 %) were lactose intolerant. Conclusion: This study suggests that lactose intolerance was significantly higher (p=0.0301) in NASH patients as compared to healthy controls. INASL-8 Nonalcoholic steatohepatitis - Correlation of clinical, biochemical and histopathological profile Mathew Vadukot, R Harikumar, Malini Eapen, V A Narayanan, V Balakrishnan Digestive Diseases Institute, Amrita Institute of Medical Sciences, AIMS Ponekkara PO, Kochi 682 041, India Background: NASH has been associated with insulin resistance, obesity, Type 2DM, dyslipidemia and HTN. Aim of the Study: Aim is to correlate various clinical, biochemical and histopathological parameters. Methods: One hundred and eighty-six patients with NAFLD presenting to the liver clinic with persistently elevated ALT more than 3 x ULN >6 months were evaluated. Out of this, 41 (34 males and 7 females) patients gave consent for liver biopsy. Apart from routine biochemistries, HOMA-IR, CRP, uric acid and metabolic profile were assessed. Histopathologically Brunt grade, Kleiner stage, and NAS scores were obtained. Statistical analysis chi-square test used for testing significance between two categorical variables. Independent sample t -test (parametric) and Mann-Whitney U test (nonparametric) were used for comparing the average of two groups. Spearman’s correlation was used for finding the degree of relation between two continuous variables; p-value of 80 g/day. Cytochrome P450 2E1 (CYP2E1) and catalase (CAT-21A>T) genes are involved in oxidative pathway of alcohol metabolism in liver. CYP2E1 and CAT might play an important role in ALD patients from North India. Therefore, this study was planned to assess the relationship of these polymorphisms with alcohol induced liver disease. Methods: Ninety-five male patients of ALD with age range 30-65 years and 100 age matched healthy subjects were enrolled in present study. Genotypic allele frequencies of CYP2E1 and CAT were determined using polymerase chain reaction and restriction fragment length polymorphism. Results: Polymorphic alleles distribution of CYP2E1 in ALD was 66.3 % c1/c1, 13.7 % c1/c2 and 20 % c2/c2 while in controls, 70 % CYP2E1*c1/c1, 5 % c1/c2 and 25 % c2/c2. Genetic polymorphic alleles of CAT 21A>T in ALD was 69.5 % AT, 25.2 % AA and 5.3 % TT while in controls, 73 % CAT AT, 10 % AA and 17 % TT. CYP2E1*c1/c2 and CAT homozygote (AA) genotype frequencies were significantly higher (p