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Hepatitis B virus genotypes in acute and fulminant hepatitis patients from north India using two different molecular genotyping approaches
Hepatology Research 35 (2006) 79–82
Short communication
Hepatitis B virus genotypes in acute and fulminant hepatitis patients from north India using two different molecular genotyping approaches Saket Chattopadhyay a , Bhudev Chandra Das b , Zahid Hussain a , Premashis Kar a,∗ a
PCR- Hepatitis Laboratory, Department of Medicine, Maulana Azad Medical College, New Delhi 110002, India b Institute of Cytology and preventive Oncology, (ICMR), Noida 201301, U.P., India Received 19 November 2005; received in revised form 4 March 2006; accepted 7 March 2006 Available online 19 April 2006
Abstract 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 < 0.01, genotype D versus A). Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (p > 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. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Acute hepatitis B; Fulminant hepatitis B; HBV genotypes; RFLP; Type-specific primer-based PCR (TSP-PCR)
1. Introduction Infection of hepatitis B virus (HBV), a member of hepadnaviridae family, is a global health problem. Approximately, 2 billion people in the world have been infected by HBV, that causes liver disease, 350 million of whom are chronic carriers of the virus [1,2]. Worldwide HBV isolates have been classified into eight genotypes, A, B, C, D, E, F, G and H [1,3]. HBV genotypes have a characteristic geographical distribution [1]. Several studies have revealed the strong association of HBV genotypes with the severity of liver disease [1,4–6]. Hepatitis B is endemic in India with an estimated national HBsAg carrier rate of 4.7% [7]. HBV has been found to be the major etiologic agent of chronic liver diseases from different parts of the country [8,9]. ∗ Correspondence to: Premashis Kar, D-II/M-2755, Netaji Nagar, New Delhi 110023, India. Fax: +91 11 232 30 132. E-mail address: [email protected] (S. Chattopadhyay).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.03.002
This is the first study from north India on the prevalence and clinical correlation of HBV genotypes prevalent in acute and fulminant hepatic failure cases using two genotyping methods based on restriction fragment length polymorphism (RFLP) [10] and polymerase chain reaction (PCR) with typespecific primers [11] both targeting to the S-gene encoding the hepatitis B surface antigen.
2. Materials and methods 2.1. Patients (1) Asymptomatic HBsAg carrier (ASC) (n = 40) with normal serum alanine amino transferase (ALT) levels for ≥1 year (<45 IU/L). (2) Acute hepatitis B (AHB) was defined as those HBV related cases which have acute self-limited disease with a serum aspartate aminotransferase (AST) eleva-
80
S. Chattopadhyay et al. / Hepatology Research 35 (2006) 79–82
tion of at least five-fold or clinical jaundice or both (n = 80). (3) Fulminant hepatitis B (FHB) was considered in those HBV related cases when after a typical acute onset, the patient became deeply jaundiced and went into hepatic encephalopathy within 4 weeks of the onset of the disease with no past history of pre-existing liver disease (n = 40). Asymptomatic carriers were identified during serosurveys conducted by Lok Nayak Hospital staff or screening associated with blood donation camps, whereas, consecutive AHB and FHB patients were admitted in different wards of Lok Nayak Hospital, New Delhi, India during the period June 2001–April 2005. The FHB patients were screened separately between the period June 2001 and April 2005. During this period, out of 200 various FH cases, 40 FHB patients were diagnosed and selected for the study. Likewise, out of 1200 acute viral hepatitis cases seen in Lok Nayak Hospital during the same period, 80 AHB patients were diagnosed and selected for the study. The study was approved by the institutional Ethical committee of Maulana Azad Medical College, New Delhi. Informed consent was obtained before patient enrollment. 2.2. Detection of HBsAg, HBeAg, IgM anti-HBc, anti-HCV and anti-HDV Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and IgM antibodies to hepatitis B core antigen (IgM anti-HBc) were investigated by third generation enzyme immuno-assay (EIA) method using commercially available EIA kits (Abbott Laboratories, Chicago, IL, USA). AntiHCV by Innotest HCV AB III (Innogenetics NV, Ghent, Belgium) and IgM anti-HDV (Abbott Laboratories) were also tested.
enzymes, i.e. EarI, AlwI, NciI, HphI and NlaIV [10]. The digested amplicons were run on 3% agarose gel stained with ethidium bromide and were observed under ultraviolet (UV) light. 2.6. Determination of HBV genotypes by PCR using type-specific primers Genotyping was done on the basis of PCR based typespecific DNA bands as described earlier [11]. 2.7. HBV genotype analysis based on nucleotide sequencing Representative PCR amplified products of S-gene (485 bases) were sequenced using the dideoxy chain termination method in an automated DNA sequencer (ABI 37718 sequencing system, Perkin-Elmer). Nucleotide sequences were aligned with CLUSTALW software. DNA sequences of genotypes A–H were downloaded from the GenBank according to the accession numbers for the respective genotype and phylogenetic analysis was carried out with the CLUSTALW Algorithm. 2.8. Statistical analysis Quantitative data was expressed as Mean ± S.D. (standard deviation). Statistical significance was determined by Chisquare test with Yates’ correction (wherever needed) or by two-sided Fisher exact test and Student’s t-test using SPSS software. The p values of less than 0.05 were considered significant throughout.
3. Results
2.3. Biochemical investigations
3.1. Demographic and clinical profile of the patients
AST, ALT, alkaline phosphatase (ALP), total bilirubin (T. Bil.), prothrombin time (PT), biochemical investigations were carried out weekly while admitted and at every month follow up after the patients were discharged from the hospital.
In all, 160 HBV DNA positive cases were included in the study for genotype analysis. The S-gene of HBV isolates from 80 AHB patients and 40 FHB patients were analysed for HBV genotypes. Also, 40 ASC were analysed for HBV genotypes which served as controls. The demographic and baseline clinical characteristics of the patients belonging to the three different groups is shown in Table 1. The mean age of ASC group was not significantly different than AHB and FHB cases. In all the three groups the frequency of male patients were more than the female patients (Table 1). None of the patients were anti-HCV or anti-HDV positive as revealed by EIA tests.
2.4. Extraction of DNA from serum and amplification of S-gene of HBV by PCR Total nucleic acid from 100 L serum was isolated using standard Proteinase-k/phenol/chloroform method [12]. Part of the S-gene was amplified by nested PCR as described earlier [10]. 2.5. Determination of HBV genotypes by RFLP analysis of S-gene The second round PCR Product with a length of 485 bp was subjected to digestion with five kinds of restriction
3.2. HBV genotyping by PCR-RFLP The PCR products of 160 HBV DNA positive subjects were digested with restriction enzymes specific for genotypes B, C, E and F and no characteristic fragment for each
S. Chattopadhyay et al. / Hepatology Research 35 (2006) 79–82 Table 1 Demographic and clinical characteristics of patients in the three study groups Parameter (n)
AHB 80
FHB 40
ASC 40
Male:female Mean age (years) ALT (IU/L) AST (IU/L) PT (T/C-s) Total Bil. (mg/dl) IgM anti-HBc (%)
56:24 (2.3:1) 36.75 ± 11.34 240.28 ± 77.69 233.77 ± 46.71 17.47 ± 3.57 12.26 ± 2.50 99
27:13 (2:1) 30.85 ± 13.47 1526 ± 278.39 1329 ± 225.58 39.2 ± 7.90c 19.57 ± 3.68d 97e
31:9 (3.4:1)a 39.70 ± 7.92b 29.5 ± 8.46 34.6 ± 5.72 N.D. 0.84 ± 0.22 0
22 5.8 9 4
18 4.1 6 7f
13 2 4 1
59.2
64.9
80
(3/80) 3.7 (77/80) 96.2 89
(2/40) 5 (38/40) 95 85
(7/40) 15 (33/40) 85g 88h
Risk factors (%) Transfusions Tattoo Operation Intravenous drug abuse Unknown Genotype A (%) Genotype D (%) HBeAg +ve (%)
AHB, acute hepatitis B; FHB, fulminant hepatitis B; ASC, asymptomatic HBsAg carrier. a ×2 , AHB vs. FHB N.S., FHB vs. ASC, AHB vs. ASC < 0.05, significant. b Mean age in AHB vs. FHB, AHB vs. ASC, FHB vs. ASC, p > 0.05, N.S. c t-Test AHB vs. FHB groups, p < 0.01, significant. d t-Test AHB vs. FHB groups, p < 0.05, significant. e t-Test AHB vs. FHB groups, p > 0.05, not significant. f t-Test AHB vs. FHB groups, p > 0.05, not significant. g t-Test genotype D vs. genotype A, in AHB, FHB and ASC groups, p < 0.01, significant. h HBeAg +ve, AHB vs. FHB, FHB vs. ASC, AHB vs. ASC > 0.05, not significant (N.S.).
genotype were observed. Finally, by digestion with NlaIV, characteristic fragments of genotype A were observed in 12 subjects, characteristic fragments of genotype D were found in the rest of the 147 subjects. However, 1 patient out of the 148 could not be genotyped by analysing the restriction pattern. 3.3. HBV genotyping by PCR with type-specific primers Twelve (7.5%) of the 160 subjects had a PCR product of 68 bp that was characteristic of genotype A. The other 148 (92.5%) out of the 160 subjects had PCR product length of 119 bp and were classified into genotype D. The patient who could not be genotyped by RFLP had a PCR product length of 119 bp and thus was classified as genotype D. 3.4. Verification of RFLP and TSP-PCR genotyping methods by sequencing Seventeen representative sequences were selected randomly from both genotypes A and D samples. The 17 sequences were recorded in GenBank (accession numbers: AY847270 and AY939894–AY939909). A phylogenetic tree was constructed based on 410 bp of the S-gene of the HBV
81
genome using CLUSTALW software. Out of the 17 isolates, 15 isolates were clustered with genotype D and 2 were clustered with genotype A. The genotype A samples were further classified into subtype A1 by blast analysis. None of the sequences were clustered with the sequences of the genotypes B, C, E, F, G or H. 3.5. Phylogenetic analysis of the indeterminate subject The sample in which genotyping by RFLP could not be established was put to nucleotide sequence based genotyping. The sequence was clustered with HBV isolates of genotype D (AY847270). 3.6. Comparison of clinical profiles between genotypes A and D in northern India Twelve (7.5%) and 148 (92.5%) of the 160 subjects were classified into genotypes A and D, respectively (Table 1). The ALT, AST levels, mean age and HbeAg, IgM anti-HBc positivity and risk factors were compared between genotypes A and D in all the study groups and no significant differences were observed. In the AHB category, infection resolved spontaneously in 77 of the 80 patients, while infection persisted in 3 (3.7%) patients all of whom were infected with genotype A of the virus. All the three patients with chronic infection were men (100%). HBsAg persisted in them during 6 months or longer after they first tested positive for it. Mean age, HBeAg positivity and mean ALT levels were not significantly different between patients in whom HBV infection did and did not persist (data not shown).
4. Discussion Although the predominance of HBV genotypes A and D has been reported in patients with HBV-related chronic liver diseases [13], we report for the first time the prevalence of HBV genotypes in acute and fulminant hepatitis patients from north India. The present study demonstrates that HBV genotype D is highly prevalent. This result, however, differs from that of an earlier study on chronic liver diseases [14] and also from east-Asian countries where genotypes B and C are found to be dominant [15–17]. The difference may be attributed to geographical variation [18]. Many simplified methods of HBV genotyping such as PCR-RFLP [10], multiplex PCR [11] and real-time PCR [1] have been used but we have attempted to validate two most commonly used methods originally described by Naito et al. and Mizokami et al. Genotypes determined by both these methods were well matched in almost all the subjects. However, one case of genotype D could not be genotyped by RFLP, and required TSP-PCR and sequencing to confirm the genotype. The 186 bp fragment, specific for HBV genotype D, was identified in this case.
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Based on the nucleotide sequence of the S-region, genotype D emerged as the predominant genotype (15/17, 88.2%). Seventeen representative samples from the study population were sequenced for the S-gene which led to confirmation of the RFLP and TSP-PCR results. Indian strains belonging to genotypes A and D differed from each other by about 5.4% in the S-gene (410 bases). In this study we did not find any mixed infection by two or more genotypes of HBV. This is in contrast to what was reported earlier [14]. On comparing the risk factors associated with transmission of HBV we found that both genotypes A and D were more often transmitted via blood transfusion mode which is in accordance with previous findings [1]. AHB with genotype A infection tends to persist and is not cleared often by anti-viral therapy [19,20]. In our study, genotype A was found only in minority of the patient categories (Table 1). Nevertheless, 3 out of 80 (3.7%) patient in AHB category who went into chronicity belonged to genotype A. The baseline characteristics of these cases were not different from other AHB cases of both genotypes A and D (p > 0.05). In this study, the rates of spontaneous recovery for patients with FHB were 57.5% and mortality rate was 42.5% (p > 0.05). The spontaneous recovery rate in our patients with FHB was worse than that of an earlier report from USA [20]. There have been no studies from northern India comparing the clinical outcome of FHB patients infected with different HBV genotypes. A significantly higher percentage of patients with FHB are infected with HBV genotype D, suggesting HBV genotype D may be associated with an increased risk of a severe course during acute infection. In conclusion, genotype D appears to be the dominant genotype circulating in north Indian population with acute and fulminant hepatitis B. Genotype A appears to be more often associated with persistent infection in comparison to genotype D which is mostly involved in severe form of fulminant hepatic failure.
References [1] Schaefer S. Hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. [2] Kao J-H. Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J Gastroenterol Hepatol 2002;17:643–50. [3] Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence comparison of surface antigen subtypes. J Gen Virol 1988;69:2575–83.
[4] Chen BF. Clinical significance of the hepatitis B virus genotypes. Fu Jen J Med 2004;2(4):273–81. [5] Mayerat C, Mantegani A, Frei PC, et al. Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection? J Viral Hepat 1999;6:299–304. [6] Tsubota A, Arase Y, Ren F, Tanaka H, Ikeda K, Kumada H. Genotype may correlate with liver carcinigenesis and tumor characteristics in cirrhotic patients infected with hepatitis B virus subtype adw. J Med Virol 2001;65:257–65. [7] Tyagarajan SP, Jayaram S, Mohanavalli B. Prevalence of HBV in the general population of India. In: Sarin SK, Singhal AK, editors. Hepatitis B in India. Problems and preventions. New Delhi, India: CBS; 1996. p. 5–16. [8] Sarin SK, Chari S, Sundaram KR, Ahuja RK, Anand BS, Broor SL. Young vs. adult cirrhotics: a prospective, comparative analysis of the clinical profile, natural course and survival. Gut 1988;29:101–7. [9] Sundaram C, Reddy CR, Ramana GV, et al. Hepatitis B surface antigen, hepatocellular carcinoma and cirrhosis in south India; an autopsy study. Indian J Pathol Microbiol 1990;33:334–8. [10] Mizokami M, Nakano T, Orita E, et al. Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns. FEBS Lett 1999;450:66–71. [11] Naito H, Hayashi S, Abe K, et al. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type specific primers. J Clin Microbiol 2001;39(1 (January)):362–4. [12] Niel C, Moraes MTB, Gaspar AMC, Yoshida CFT, Gomes SA. Genetic diversity of hepatitis B virus isolated in Rio de Janeiro, Brazil. J Med Virol 1994;44:180–6. [13] Kumar A, Kumar SI, Pandey R, Naik S, Aggarwal R. Hepatitis B virus genotype A is more often associated with severe liver disease in northern India than is genotype D. Indian J Gastroenterol 2005;24–31:19–22. [14] Thakur V, Guptan RC, Kazim SN, et al. Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent. J Gastroenterol Hepatol 2002;17:165–70. [15] Orito E, Ichida T, Sakugawa H, et al. Geographical distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 2001;34:590–4. [16] Liu CJ, Kao JH, Lai MY, et al. Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminent or subfulminant hepatitis. J Med Virol 2004;72:545– 50. [17] Yusar R, Takahashi K, Dien BV, et al. Properties of hepatitis B virus genome recovered from vietnamese patients with fulminant hepatitis in comparison with those of acute hepatitis. J Med Virol 2000;61:23–8. [18] Chu CJ, Lok AS. Clinical significance of hepatitis B virus genotypes. Hepatology 2002;35(5):1274–6. [19] Suzuki Y, Kobayashi M, Ikeda K, et al. Persistence of acute infection with hepatitis B virus genotype A and treatment in Japan. J Med Virol 2005;76:33–9. [20] Wai CT, Fontana RJ, Polson J, et al. Clinical outcome and virological characteristics of hepatitis B related acute liver failure in the United States. J Viral Hepat 2005;12:192–8.
Short communication
Hepatitis B virus genotypes in acute and fulminant hepatitis patients from north India using two different molecular genotyping approaches Saket Chattopadhyay a , Bhudev Chandra Das b , Zahid Hussain a , Premashis Kar a,∗ a
PCR- Hepatitis Laboratory, Department of Medicine, Maulana Azad Medical College, New Delhi 110002, India b Institute of Cytology and preventive Oncology, (ICMR), Noida 201301, U.P., India Received 19 November 2005; received in revised form 4 March 2006; accepted 7 March 2006 Available online 19 April 2006
Abstract 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 < 0.01, genotype D versus A). Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (p > 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. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Acute hepatitis B; Fulminant hepatitis B; HBV genotypes; RFLP; Type-specific primer-based PCR (TSP-PCR)
1. Introduction Infection of hepatitis B virus (HBV), a member of hepadnaviridae family, is a global health problem. Approximately, 2 billion people in the world have been infected by HBV, that causes liver disease, 350 million of whom are chronic carriers of the virus [1,2]. Worldwide HBV isolates have been classified into eight genotypes, A, B, C, D, E, F, G and H [1,3]. HBV genotypes have a characteristic geographical distribution [1]. Several studies have revealed the strong association of HBV genotypes with the severity of liver disease [1,4–6]. Hepatitis B is endemic in India with an estimated national HBsAg carrier rate of 4.7% [7]. HBV has been found to be the major etiologic agent of chronic liver diseases from different parts of the country [8,9]. ∗ Correspondence to: Premashis Kar, D-II/M-2755, Netaji Nagar, New Delhi 110023, India. Fax: +91 11 232 30 132. E-mail address: [email protected] (S. Chattopadhyay).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.03.002
This is the first study from north India on the prevalence and clinical correlation of HBV genotypes prevalent in acute and fulminant hepatic failure cases using two genotyping methods based on restriction fragment length polymorphism (RFLP) [10] and polymerase chain reaction (PCR) with typespecific primers [11] both targeting to the S-gene encoding the hepatitis B surface antigen.
2. Materials and methods 2.1. Patients (1) Asymptomatic HBsAg carrier (ASC) (n = 40) with normal serum alanine amino transferase (ALT) levels for ≥1 year (<45 IU/L). (2) Acute hepatitis B (AHB) was defined as those HBV related cases which have acute self-limited disease with a serum aspartate aminotransferase (AST) eleva-
80
S. Chattopadhyay et al. / Hepatology Research 35 (2006) 79–82
tion of at least five-fold or clinical jaundice or both (n = 80). (3) Fulminant hepatitis B (FHB) was considered in those HBV related cases when after a typical acute onset, the patient became deeply jaundiced and went into hepatic encephalopathy within 4 weeks of the onset of the disease with no past history of pre-existing liver disease (n = 40). Asymptomatic carriers were identified during serosurveys conducted by Lok Nayak Hospital staff or screening associated with blood donation camps, whereas, consecutive AHB and FHB patients were admitted in different wards of Lok Nayak Hospital, New Delhi, India during the period June 2001–April 2005. The FHB patients were screened separately between the period June 2001 and April 2005. During this period, out of 200 various FH cases, 40 FHB patients were diagnosed and selected for the study. Likewise, out of 1200 acute viral hepatitis cases seen in Lok Nayak Hospital during the same period, 80 AHB patients were diagnosed and selected for the study. The study was approved by the institutional Ethical committee of Maulana Azad Medical College, New Delhi. Informed consent was obtained before patient enrollment. 2.2. Detection of HBsAg, HBeAg, IgM anti-HBc, anti-HCV and anti-HDV Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and IgM antibodies to hepatitis B core antigen (IgM anti-HBc) were investigated by third generation enzyme immuno-assay (EIA) method using commercially available EIA kits (Abbott Laboratories, Chicago, IL, USA). AntiHCV by Innotest HCV AB III (Innogenetics NV, Ghent, Belgium) and IgM anti-HDV (Abbott Laboratories) were also tested.
enzymes, i.e. EarI, AlwI, NciI, HphI and NlaIV [10]. The digested amplicons were run on 3% agarose gel stained with ethidium bromide and were observed under ultraviolet (UV) light. 2.6. Determination of HBV genotypes by PCR using type-specific primers Genotyping was done on the basis of PCR based typespecific DNA bands as described earlier [11]. 2.7. HBV genotype analysis based on nucleotide sequencing Representative PCR amplified products of S-gene (485 bases) were sequenced using the dideoxy chain termination method in an automated DNA sequencer (ABI 37718 sequencing system, Perkin-Elmer). Nucleotide sequences were aligned with CLUSTALW software. DNA sequences of genotypes A–H were downloaded from the GenBank according to the accession numbers for the respective genotype and phylogenetic analysis was carried out with the CLUSTALW Algorithm. 2.8. Statistical analysis Quantitative data was expressed as Mean ± S.D. (standard deviation). Statistical significance was determined by Chisquare test with Yates’ correction (wherever needed) or by two-sided Fisher exact test and Student’s t-test using SPSS software. The p values of less than 0.05 were considered significant throughout.
3. Results
2.3. Biochemical investigations
3.1. Demographic and clinical profile of the patients
AST, ALT, alkaline phosphatase (ALP), total bilirubin (T. Bil.), prothrombin time (PT), biochemical investigations were carried out weekly while admitted and at every month follow up after the patients were discharged from the hospital.
In all, 160 HBV DNA positive cases were included in the study for genotype analysis. The S-gene of HBV isolates from 80 AHB patients and 40 FHB patients were analysed for HBV genotypes. Also, 40 ASC were analysed for HBV genotypes which served as controls. The demographic and baseline clinical characteristics of the patients belonging to the three different groups is shown in Table 1. The mean age of ASC group was not significantly different than AHB and FHB cases. In all the three groups the frequency of male patients were more than the female patients (Table 1). None of the patients were anti-HCV or anti-HDV positive as revealed by EIA tests.
2.4. Extraction of DNA from serum and amplification of S-gene of HBV by PCR Total nucleic acid from 100 L serum was isolated using standard Proteinase-k/phenol/chloroform method [12]. Part of the S-gene was amplified by nested PCR as described earlier [10]. 2.5. Determination of HBV genotypes by RFLP analysis of S-gene The second round PCR Product with a length of 485 bp was subjected to digestion with five kinds of restriction
3.2. HBV genotyping by PCR-RFLP The PCR products of 160 HBV DNA positive subjects were digested with restriction enzymes specific for genotypes B, C, E and F and no characteristic fragment for each
S. Chattopadhyay et al. / Hepatology Research 35 (2006) 79–82 Table 1 Demographic and clinical characteristics of patients in the three study groups Parameter (n)
AHB 80
FHB 40
ASC 40
Male:female Mean age (years) ALT (IU/L) AST (IU/L) PT (T/C-s) Total Bil. (mg/dl) IgM anti-HBc (%)
56:24 (2.3:1) 36.75 ± 11.34 240.28 ± 77.69 233.77 ± 46.71 17.47 ± 3.57 12.26 ± 2.50 99
27:13 (2:1) 30.85 ± 13.47 1526 ± 278.39 1329 ± 225.58 39.2 ± 7.90c 19.57 ± 3.68d 97e
31:9 (3.4:1)a 39.70 ± 7.92b 29.5 ± 8.46 34.6 ± 5.72 N.D. 0.84 ± 0.22 0
22 5.8 9 4
18 4.1 6 7f
13 2 4 1
59.2
64.9
80
(3/80) 3.7 (77/80) 96.2 89
(2/40) 5 (38/40) 95 85
(7/40) 15 (33/40) 85g 88h
Risk factors (%) Transfusions Tattoo Operation Intravenous drug abuse Unknown Genotype A (%) Genotype D (%) HBeAg +ve (%)
AHB, acute hepatitis B; FHB, fulminant hepatitis B; ASC, asymptomatic HBsAg carrier. a ×2 , AHB vs. FHB N.S., FHB vs. ASC, AHB vs. ASC < 0.05, significant. b Mean age in AHB vs. FHB, AHB vs. ASC, FHB vs. ASC, p > 0.05, N.S. c t-Test AHB vs. FHB groups, p < 0.01, significant. d t-Test AHB vs. FHB groups, p < 0.05, significant. e t-Test AHB vs. FHB groups, p > 0.05, not significant. f t-Test AHB vs. FHB groups, p > 0.05, not significant. g t-Test genotype D vs. genotype A, in AHB, FHB and ASC groups, p < 0.01, significant. h HBeAg +ve, AHB vs. FHB, FHB vs. ASC, AHB vs. ASC > 0.05, not significant (N.S.).
genotype were observed. Finally, by digestion with NlaIV, characteristic fragments of genotype A were observed in 12 subjects, characteristic fragments of genotype D were found in the rest of the 147 subjects. However, 1 patient out of the 148 could not be genotyped by analysing the restriction pattern. 3.3. HBV genotyping by PCR with type-specific primers Twelve (7.5%) of the 160 subjects had a PCR product of 68 bp that was characteristic of genotype A. The other 148 (92.5%) out of the 160 subjects had PCR product length of 119 bp and were classified into genotype D. The patient who could not be genotyped by RFLP had a PCR product length of 119 bp and thus was classified as genotype D. 3.4. Verification of RFLP and TSP-PCR genotyping methods by sequencing Seventeen representative sequences were selected randomly from both genotypes A and D samples. The 17 sequences were recorded in GenBank (accession numbers: AY847270 and AY939894–AY939909). A phylogenetic tree was constructed based on 410 bp of the S-gene of the HBV
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genome using CLUSTALW software. Out of the 17 isolates, 15 isolates were clustered with genotype D and 2 were clustered with genotype A. The genotype A samples were further classified into subtype A1 by blast analysis. None of the sequences were clustered with the sequences of the genotypes B, C, E, F, G or H. 3.5. Phylogenetic analysis of the indeterminate subject The sample in which genotyping by RFLP could not be established was put to nucleotide sequence based genotyping. The sequence was clustered with HBV isolates of genotype D (AY847270). 3.6. Comparison of clinical profiles between genotypes A and D in northern India Twelve (7.5%) and 148 (92.5%) of the 160 subjects were classified into genotypes A and D, respectively (Table 1). The ALT, AST levels, mean age and HbeAg, IgM anti-HBc positivity and risk factors were compared between genotypes A and D in all the study groups and no significant differences were observed. In the AHB category, infection resolved spontaneously in 77 of the 80 patients, while infection persisted in 3 (3.7%) patients all of whom were infected with genotype A of the virus. All the three patients with chronic infection were men (100%). HBsAg persisted in them during 6 months or longer after they first tested positive for it. Mean age, HBeAg positivity and mean ALT levels were not significantly different between patients in whom HBV infection did and did not persist (data not shown).
4. Discussion Although the predominance of HBV genotypes A and D has been reported in patients with HBV-related chronic liver diseases [13], we report for the first time the prevalence of HBV genotypes in acute and fulminant hepatitis patients from north India. The present study demonstrates that HBV genotype D is highly prevalent. This result, however, differs from that of an earlier study on chronic liver diseases [14] and also from east-Asian countries where genotypes B and C are found to be dominant [15–17]. The difference may be attributed to geographical variation [18]. Many simplified methods of HBV genotyping such as PCR-RFLP [10], multiplex PCR [11] and real-time PCR [1] have been used but we have attempted to validate two most commonly used methods originally described by Naito et al. and Mizokami et al. Genotypes determined by both these methods were well matched in almost all the subjects. However, one case of genotype D could not be genotyped by RFLP, and required TSP-PCR and sequencing to confirm the genotype. The 186 bp fragment, specific for HBV genotype D, was identified in this case.
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Based on the nucleotide sequence of the S-region, genotype D emerged as the predominant genotype (15/17, 88.2%). Seventeen representative samples from the study population were sequenced for the S-gene which led to confirmation of the RFLP and TSP-PCR results. Indian strains belonging to genotypes A and D differed from each other by about 5.4% in the S-gene (410 bases). In this study we did not find any mixed infection by two or more genotypes of HBV. This is in contrast to what was reported earlier [14]. On comparing the risk factors associated with transmission of HBV we found that both genotypes A and D were more often transmitted via blood transfusion mode which is in accordance with previous findings [1]. AHB with genotype A infection tends to persist and is not cleared often by anti-viral therapy [19,20]. In our study, genotype A was found only in minority of the patient categories (Table 1). Nevertheless, 3 out of 80 (3.7%) patient in AHB category who went into chronicity belonged to genotype A. The baseline characteristics of these cases were not different from other AHB cases of both genotypes A and D (p > 0.05). In this study, the rates of spontaneous recovery for patients with FHB were 57.5% and mortality rate was 42.5% (p > 0.05). The spontaneous recovery rate in our patients with FHB was worse than that of an earlier report from USA [20]. There have been no studies from northern India comparing the clinical outcome of FHB patients infected with different HBV genotypes. A significantly higher percentage of patients with FHB are infected with HBV genotype D, suggesting HBV genotype D may be associated with an increased risk of a severe course during acute infection. In conclusion, genotype D appears to be the dominant genotype circulating in north Indian population with acute and fulminant hepatitis B. Genotype A appears to be more often associated with persistent infection in comparison to genotype D which is mostly involved in severe form of fulminant hepatic failure.
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