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ESPS Manuscript NO: 3715
Columns: ORIGINAL ARTICLE
Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India
Gopalakrishnan D et al. Hepatitis B virus infection in Kerala
Deepak Gopalakrishnan, Mark Keyter, Kotacherry Trivikrama Shenoy, Leena Kondarappassery Balakumaran, Thayumanavan Barathi, Varghese Thomas, KR Vinayakumar, Charles Panackel, Arun T Korah, Ramesh Nair, Anna Kramvis
Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa
Deepak Gopalakrishnan, KR Vinayakumar, Charles Panackel, Arun T Korah, Ramesh Nair, Department of Gastroenterology, Medical College Trivandrum 695011, India
Kotacherry Trivikrama Shenoy, Leena Kondarappassery Balakumaran, Population Health and Research Institute, Trivandrum 695011, India
Thayumanavan Barathi, Government Rajaji Hospital, Madurai Medical College, Madurai 625002, India
Varghese Thomas, Department of Gastroenterology, Calicut Medical College, PO Calicut 673008, India
Author contributions: Gopalakrishnan D and Keyter M contributed equally to the study; Gopalakrishnan D, Shenoy KT, Kramvis A conceptualized the study; Gopalakrishnan D, Shenoy KT, Balakumaran LK, Barathi T, Thomas V, Vinayakumar KR, Panackel C, Korah AT, Nair R collected the clinical data; Gopalakrishnan D, Keyter M and Kramvis A conceived and designed the laboratory experiments; Kramvis A contributed reagents/materials/analysis tools; Gopalakrishnan D and Keyter M performed the experiments; Gopalakrishnan D, Keyter M and Kramvis A analyzed and interpreted the data, wrote the paper.
Supported by National Research Foundation of South Africa, NRF; GUN 65530 (to Kramvis A) and Cancer Association of South Africa; Postdoctoral Funding from the NRF, GUN 75055, University of the Witwatersrand (to Gopalakrishnan D); Bursaries from the University of the Witwatersrand, the Poliomyelitis Research Foundation and the Ernst and Ethel Eriksen Trust (to Keyter M)
Correspondence to: Anna Kramvis, Professor, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa. HYPERLINK "mailto:anna.kramvis@wits.ac.za" anna.kramvis@wits.ac.za
Telephone: +27-11-4883100 Fax: +27-86-5296806
Received: May 16, 2013 Revised: June 20, 2013
Accepted: July 17, 2013
Published online:
Abstract
AIM: To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.
METHODS: Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually and compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. TaqMan BS-1 probe was used to quantify HBV DNA with a l o w e r d e t e c t i o n l i m i t o f a p p r o x i m a t e l y 2 0 I U / m L . C o n t i n u o u s v a r i a b l e s w e r e c o m p a r e d u s i n g a n i n d e p e n d e n t S t u d e n t s t t e s t . T h e 2 t e s t o r F i s h e r s e x a c t t e s t w a s u s e d t o c o m p a r e c a t e g o r i c a l v a r i a b l e s . T h e d i f f e r e n c e s w e r e c o n s i d e r e d s t a t i s t i c a l l y s i g n i f i c ant for P < 0.05.
RESULTS: Irrespective of disease status, the predominant genotype was A (72%), 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. A1762T/G1764A was significantly associated with HCC in both genotypes A and D. G1862T was more frequent in subgenotype A1 (P < 0.0001) and in combination with A1762T/G1764A was significantly associated with HBV from HCC patients. C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The pre-S2 start codon M1T/I mutation, was unique to genotype A strains (15.6%), from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of pre-S deletion mutants (33.3%), was observed in genotype A from HCC, compared to non-HCC patients, but did not reach statistical significance. preS2: F22L was found in genotypes A and D.
CONCLUSION: Kerala is the first Indian state, where subgenotype A1 was found to predominate in liver disease patients with the development of HCC at a relatively young age.
2013 Baishideng. All rights reserved.
Key words: Hepatocellular carcinoma; Cirrhosis; Chronic hepatitis; Phylogenetic analysis; Genotype; India
Core tip: This study shows the predominance of subgenotype A1 in liver disease patients in Kerala, and its high prevalence in hepatocellular carcinoma (HCC) patients. Subgenotype A1 could be more hepatocarcinogenic and HCC develops at a younger age, irrespective of host ethnicity. The S open reading frame of subgenotype A1 isolates from Kerala clustered separately within the Asian clade and encoded distinct subgenotype A1 amino acids. A higher frequency of G1862T was detected compared to subgenotype A1 isolates from other geographical regions. This is the first time that pre-S deletion mutants have been described in Indian HCC patients.
Gopalakrishnan D, Keyter M, Shenoy KT, Balakumaran LK, Barathi T, Thomas V, Vinayakumar KR, Panackel C, Korah AT, Nair R, Kramvis A. Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India. World J Gastroenterol 2013;
Available from: URL: http://www.wjgnet.com/esps/
DOI: http://dx.doi.org/10.3748/wjg.v19.i0.0000
INTRODUCTION
Hepatitis B virus (HBV) is the prototype member of the family HePadnaviridae. HBV replicates by reverse transcription using a polymerase, lacking proof reading ability, and sequence heterogeneity is a feature of this virus. Phylogenetic analysis of HBV full-length genomes has led to the classification of HBV into nine genotypes (AI), defined by an intergroup divergence in the complete HBV genome sequence of 7.5% or more and a tenth genotype J, which was found in a single individual, has been proposed ADDIN EN.CITE ADDIN EN.CITE.DATA [1]. Genotypes A, B, C, D, F and I are further classified into subgenotypes. Most genotypes and some subgenotypes display distinct geographical distribution. Moreover, HBV genotypes and, in some cases, subgenotypes have been shown to play an important role in the clinical consequences of the infection as well as in the response to antiviral treatment.
HBV infection remains a significant global health problem with an estimated two billion people infected and more than 240 million are chronic carriers of the virus, leading to 600000 deaths from the clinical consequences of infection, including cirrhosis, liver failure and hepatocellular carcinoma (HCC). With a population of more than 1.2 billion people, India has the second largest global pool of chronic HBV infection and HBV is the major cause of liver disease in India ADDIN EN.CITE Acharya200621472147214717Acharya, S. K.Madan, K.Dattagupta, S.Panda, S. K.Department of Gastroenterology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. subratacharya2004@yahoo.comViral hepatitis in IndiaNatl Med J IndiaNatl Med J India203-171942006/11/15Carcinoma, Hepatocellular/etiologyCost of IllnessHepatitis A/epidemiology/prevention & control/virologyHepatitis B/drug therapy/epidemiology/transmissionHepatitis C/drug therapy/epidemiology/virologyHepatitis E/epidemiology/prevention & control/virology*Hepatitis, Viral, Human/drug therapy/epidemiology/virologyHumansIndiaLiver Neoplasms/etiologyPrevalence2006Jul-Aug0970-258X (Print)
0970-258X (Linking)17100109http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17100109eng[2].
Most studies have estimated hepatitis B surface antigen (HBsAg) carrier rate to be between 2% and 8%, placing India within the zone of intermediate endemicity. A HBsAg prevalence rate of 2.97% was found among the rural population ADDIN EN.CITE ADDIN EN.CITE.DATA [3], and a meta-analysis has reported the mean prevalence in the general population of India as 3.3% ADDIN EN.CITE Thyagarajan SP200036953695369517Thyagarajan SP, Hari R, Murugavel KG. Prevalence of hepatitis B virus infection in general population of India.Ind J GastroenterolInd J Gastroenterol19(suppl 3):C102000[4]. However, these estimates have been questioned because, according to Phadke and Kale (2002), the often quoted estimate for India of 4.7% was obtained by incorrectly pooling results of a set of studies including unrepresentative high risk groups and also equating the HYPERLINK "http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1444854/" \o "Click to Continue > by Text-Enhance"single test HBsAg positivity rate with the carrier rate. By correcting for these errors, they estimated a carrier rate of 1.4% ADDIN EN.CITE Phadke200221422142214217Phadke, AnantKale, AshokHBV carrier rate in IndiaIndian pediatricsIndian pediatrics787-7873982002INDIAN PEDIATRIC0019-6061[5].
The known HBV genotype distribution in India is summarized in Figure 1. Overall, at approximately 65%, genotype D predominates, being the dominant genotype in Delhi in the north, Pune in the west and the Nicobar Islands in the south. Genotype A has been found in approximately 30%, with the highest frequency found in northern India. At approximately 5%, genotype C is found in the minority, with highest frequency in eastern and southern India. The subgenotypes that have been described in India include A1, A2, C1, C2, D1, D2, D3, D5 and D9 ADDIN EN.CITE ADDIN EN.CITE.DATA [6-9].
Kerala is the most densely populated state of India, with a population of 33 million. HBsAg prevalence of 0.5% in the normal population has been reported in northern Kerala ADDIN EN.CITE Sandesh200636973697369717Sandesh, K.Varghese, T.Harikumar, R.Beena, P.Sasidharan, V. P.Bindu, C. S.Tony, J.Harish, K.Sunilkumar, K.Ramachandran, T. M.Department of Gastroenterology, Medical College, Calicut 673 008, Kerala. drsandeshk@gmail.comPrevalence of Hepatitis B and C in the normal population and high risk groups in north KeralaTrop GastroenterolTrop Gastroenterol80-32722006/11/09AdultFemaleHepatitis B/diagnosis/ epidemiologyHepatitis B Surface Antigens/bloodHepatitis C/diagnosis/ epidemiologyHepatitis C Antibodies/bloodHumansIndia/epidemiologyMaleMiddle AgedPrevalenceRisk FactorsSeroepidemiologic Studies2006Apr-Jun0250-636X (Print)
0250-636X (Linking)17089617Nlmeng[10] and a HBsAg prevalence of 1.5%, was detected among voluntary blood donors from Trivandrum, South Kerala ADDIN EN.CITE Anjali201221462146214617Anjali, H.Issac, A.Anjali, M. R.Anish, T. S.Department of Community Medicine, Government Medical College, Thiruvananthapuram, Kerala, India.Transfusion-transmissible infections among voluntary blood donors at Government Medical College Thiruvananthapuram, Kerala, IndiaAsian J Transfus SciAsian J Transfus Sci55-6612012/05/252012Jan1998-3565 (Electronic)
0973-6247 (Linking)22623852335363910.4103/0973-6247.95060 [doi]
AJTS-6-55 [pii]Nlmeng[11]. There is a paucity of information, on the prevalence of HBV genotypes and the respective subgenotypes in Kerala, as well as their association if any, with different clinical manifestations following infection with HBV. We investigated the distribution of HBV genotypes/subgenotypes among patients with different clinical manifestations of HBV infection and molecularly characterized the viral isolates.
MATERIALS AND METHODS
Patients
The cross-sectional study was conducted from January 2005 to December 2009 during which sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV related HCC from Medical College Trivandrum, Kerala, India. The serum samples were stored at -80C until use. Serum alanine transaminase (ALT) level of < 10 times the upper limit of normal (U L N ) , s e r u m b i l i r u b i n l e v e l s o f l e s s t h a n 2 . 5 t i m e s o f U L N a n d d e t e c t a b l e H B s A g f o r e" 6 m o w e r e u s e d a s i n c l u s i o n c r i t e r i a . T h e p r e s e n c e o f h e p a t i t i s B e A n t i g e n ( H B e A g ) w a s e x a m i n e d a t t h e t i m e o f s c r e e n i n g . A l l p a t i e n t s w e r e n e g a t i v e f o r a n t i b o d i e s t o h e patitis C virus, hepatitis D virus and human immunodeficiency virus. The study protocol conformed to the 1975 Declaration of Helsinki and was approved by the ethics committees of the Medical College Trivandrum, India and the University of the Witwatersrand, South Africa.
The diagnosis of HBV related liver disease was based on clinical data, laboratory tests, liver biopsy and imaging studies and patients were classified into three groups: Group-I (HCC): The 44 patients with HCC were diagnosed by ultrasoun d s c a n a n d e l e v a t e d s e r u m f e t o p r o t e i n l e v e l s ( e" 4 0 0 n g / m L ) a n d t h e p r e s e n c e o f a l e s i o n o f e" 5 c m . G r o u p - I I ( C R - C i r r h o s i s ) : 2 2 p a t i e n t s , w i t h n e c r o - i n f l a m m a t o r y d a m a g e , f i b r o s i s w i t h n o d u l e f o r m a t i o n c o n f i r m e d b y l i v e r b i o p s y , w i t h u l t r a s o n o g r a p h i c e v i d e n c e o f p o r t a l h y p e r t e n s i o n . G r o u p - I I I ( C H - C h r o n i c H e p a t i t i s ) : 2 5 p a t i e n t s , w i t h H B s A g p o s i t i v e s t a t u s f o r e" 6 m o w i t h n o r m a l o r i n t e r m i t t e n t l y e l e v a t e d A L T ( 1 . 5 t i m e s u p p e r l i m i t o f n o r m a l ) . P a t i e n t s i n t h i s g r o u p w e r e c o n s i d e r e d f o r l i v e r b i o p s y o n t h e b asis of elevated ALT levels and HBeAg-status, and diagnosed with cirrhosis using Histological Activity index (HAI) and Fibrosis scores.
Serologic assays
All serum samples were screened for HBsAg and HBeAg using ELISA kits (DiaSorin S.P.A, Italy) according to the manufacturers instructions. Laboratory evaluation included routine liver biochemistry ALT and aspartate transaminase levels, total bilirubin, albumin, alkaline phosphatase, total protein and prothrombin time). Liver function tests were performed to find necro-inflammatory activity using Hitachi 902 Fully Automated Chemistry Analyzer (Japan). The upper limit of normal (ULN) of ALT 40 IU/L was used for diagnosis.
Real-time polymerase chain reaction quantification of HBV DNA
Polymerase chain reaction (PCR) primers, HBV-Taq1 and HBV-Taq2 covering a region of the S gene (321 to 401 from the EcoRI site) with a FAM/TAMRA labeled TaqMan BS-1 probe were used to quantify HBV DNA in an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, United States). The second WHO International Standard for HBV Nucleic Acid Amplification Techniques (product code 97/750 National Institute for Biological Standards and Control; Hertfordshire, United Kingdom), which has a final concentration of 106 IU/mL, was used as the internal standard. The lower detection limit of our assay is approximately 20 IU/mL. The conversion formula of IU = copies/4.7 was used ADDIN EN.CITE ADDIN EN.CITE.DATA [12].
PCR and restriction fragment length polymorphism assay for g e n o t y p i n g a n d m o l e c u l a r c h a r a c t e r i z a t i o n
T o t a l H B V D N A w a s e x t r a c t e d f r o m s e r u m u s i n g Q I A a m p D N A B l o o d M i n i k i t ( Q I A G E N G m b H , H i l d e n , G e r m a n y ) , a c c o r d i n g t o t h e m a n u f a c t u r e r s i n s t r u c t i o n s . T h e c o m p l e t e S O R F w a s a m p l i f i e d u s i n g a n e s t e d P C R .
P r i m e r s S 1 F 5 2 - C A A T C G C C G C G T C G C A G A A G A T C T C A A T C - 3 2 ( 2 4 1 0 - 2 4 3 9 f r o m E c o R I s i t e ) a n d S 1 R 5 2 - T C C A G A C C X G C T G C G A G C A A A A C A - 3 2 ( 1 3 1 4 - 1 2 9 1 f r o m E c o R I s i t e ) w e r e u s e d f o r t h e f i r s t r o u n d a n d S 2 F 5 2 - A A T G T T A G T A T T C C T T G G A C T C A T A A G G T G G G - 3 2 ( 2 4 5 1 - 2 4 8 2 f r o m E c o R I s i t e ) a n d S 2 R 5 2 - A G T T C C G C A G T A T G G A T C G G C A G A G G A - 3 2 ( 1 2 8 0 - 1 2 5 4 f r o m E c o R I s i t e ) f o r t h e s e c o n d r o u n d P C R u s i n g r e a c t i o n c o n d i t i o n s t h a t w a s p r e v i o u s l y r e p o r t e d . A D D I N E N . C I T E <