Aim: To determine the prevalence of hepatitis B virus (HBV) genotypes in Iranian hepatitis B surface antigen (HBsAg) carriers, chronic hepatitis B and cirrhotic patients. Methods: A total of 109 HBsAg-positive patients were included in this study. HBV genotypes were determined by using INNO-LiPA methodology which is based on the reverse hybridization principle. Results: The distribution of patients with different stages of liver disease was as follows: 95 (86.4) chronic hepatitis, 11 (10) liver cirrhosis, and 3 (2.7) inactive carrier. Of the chronic hepatitis and liver cirrhosis patients, 26.4 were HBeAg-positive while 70 were HBeAg-negative. Genotype D was the only detected type found in all patients. Conclusion: Classifying HBV into genotypes has to be cost-effective and clinically relevant. Our study indicates that HBV genotype D prevails in the Mediterranean area, Near and Middle East, and South Asia. Continued efforts for understanding HBV genotype through international co-operation will reveal further virological diffierences of the genotypes and their clinical relevance. Â© 2006 The WJG Press. All rights reserved

Alavian, S.-M.

Ashayeri, N.

Keyvani, H.

Rezai, M.

Sadeghi, H.M.

English

PubMed

eprints Iran University of Medical Sciences

Alavian SM, Keyvani H, Rezaei M, Ashayeri N, Sadeghi HM. Preliminary report of HBV genotype prevalence in Iran. World J Gastroenterol 2006; 12(32): 5211-5213 http://www.wjgnet.com/1007-9327/12/5211.aspINTRODUCTIONHuman hepatitis B virus (HBV), which is the prototype member of  the family Hepadnaviridae, is a circular, partially double-stranded DNA virus of  approximately 3200 nt[1]. This highly compact genome contains the four major open reading frames encoding the envelope (preS1, preS2 and surface antigen HBsAg), polymerase (HBPol) and X (HBX) proteins, respectively[2]. HBV is an etiologic agent of  acute and chronic liver disease, including fatal fulminant hepatitis, liver cirrhosis and hepatocellular carcinoma[3-6]. Over 2 billion people worldwide have been exposed to HBV and 350 million are chronic carriers of  HBV[7-9].In 1988, HBV was classified into four genotypes by a sequence divergence in the entire genome exceeding 8%, and designated by capital letters of  the alphabet from A to D[10,11]. In 1994, Norder et al[12] found an additional two HBV genotypes by the same criteria, and named them E and F, respectively. Genotype G was reported recently in 2000[2] and genotype H, which is phylogenetically closely related to genotype F, was proposed in 2002[11]. HBV genotypes have distinct geographical distribution[2,7,13,14].In general , genotype A is pandemic, but most prevalent in North West Europe, North America, Central Africa[2,13] and India[7]. Genotypes B and C are prevalent in Asia[7,8,13,15], especially in populations of  Eastern Asia and the far East origin[3,16]. Genotype D is also more or less pandemic, but is predominant in the Mediterranean area and the Middle East[2,3,16]. Genotype E is restricted to Africa and genotype F is found in Central and South America[7,8,13,16]. Genotype G has been recently identified in France and North America[7,8]. It has been reported that there are remarkable differences in the clinical and virologic characteristics between the patients with different genotypes[17,18,19]. According to Iranian studies[20], over 35% of  Iranians have been exposed to HBV, approximately 2% are chronic carriers. Compared to the United States where HBV infection is responsible for 25% of  chronic hepatitis, HBV accounts for up to 70%-80% of  chronic hepatitis cases in Iran, indicating that HBV alone is the leading  RAPID COMMUNICATIONPreliminary report of hepatitis B virus genotype prevalence in IranSeyed-Moayed Alavian, Hossein Keyvani, Mahdi Rezai, Neda Ashayeri, Homa Mohammad Sadeghiwww.wjgnet.comSeyed-Moayed Alavian, Department of Internal Medicine, Baqiatollah Medical University, Tehran Hepatitis Center, 92 Vesal Shirazi Street, Boolvar Keshavarz, PO Box 14155/3651, Tehran, IranHossein Keyvani, Department of Virology, Iran University of Medical Sciences, Hemmat Expressway, Tehran, IranMahdi Rezaei, Neda Ashayeri, Homa Mohammad Sadeghi, Student’s Research Committee, Iran University of Medical Sciences, Hemmat Expressway, Tehran, Iran   Supported by Tehran Hepatitis Center, Keivan Virology LabCo-first-author: Seyed-Moayed AlavianCo-correspondence: Hossein KeivaniCorrespondence to: Dr. Seyed-Moayed Alavian, Associate Professor of Gastroenterology, Department of Internal Medicine, Baqiatollah Medical University, Tehran Hepatitis Center, Iran. manager@iranhepgroup.infoTelephone: +98-21-8967923Received: 2004-11-30                Accepted: 2005-01-05AbstractAIM: To determine the prevalence of hepatitis B virus (HBV) genotypes in Iranian hepatitis B surface antigen (HBsAg) carriers, chronic hepatitis B and cirrhotic patients.METHODS: A total of 109 HBsAg-positive patients were included in this study. HBV genotypes were determined by using INNO-LiPA methodology which is based on the reverse hybridization principle. RESULTS: The distribution of patients with different stages of liver disease was as follows: 95 (86.4%) chronic hepatitis, 11 (10%) liver cirrhosis, and 3 (2.7%) inactive carrier. Of the chronic hepatitis and liver cirrhosis patients, 26.4% were HBeAg-positive while 70% were HBeAg-negative. Genotype D was the only detected type found in all patients.CONCLUSION: Classifying HBV into genotypes has to be cost-effective and clinically relevant. Our study indicates that HBV genotype D prevails in the Mediterranean area, Near and Middle East, and South Asia. Continued efforts for understanding HBV genotype through international co-operation will reveal further virological differences of the genotypes and their clinical relevance.© 2006 The WJG Press. All rights reserved.Key words: Hepatitis B virus; Genotype; Chronic Hepatitis B; CirrhosisPO Box 2345, Beijing 100023, China                                                                                                                  World J Gastroenterol  2006 August 28; 12(32): 5211-5213www.wjgnet.com                                                                                                                                          World Journal of Gastroenterology  ISSN 1007-9327wjg@wjgnet.com                                                                                                                                                                       © 2006 The WJG Press. All rights reserved.cause of  chronic liver disease in Iran[21]. Until now, to our knowledge, no data regarding HBV genotype is available, and also the genotypes distribution of  HBV and genotype-related differences with the liver disease are still unclear in Iran. In this study, we therefore determined the prevalence of  HBV genotypes in Iranian hepatitis B surface antigen (HBsAg) carriers, chronic hepatitis B and cirrhotic patients.MATERIALS AND METHODSThis cross-sectional study was performed in Tehran Hepatitis Center in 2004. A total of  109 patients with hepatitis B surface antigen (HBsAg) positive for at least 6 mo were enrolled in this study. Of  the 109 patients, 95 were classified as chronic hepatitis, defined as HBsAg positivite with or without the presence of  HBeAg, high level of  HBV-DNA (> 100 000 copies/mL) detected by Amplicor HBV monitor, persistent or intermittent elevation in ALT levels and compatible liver biopsy. Three were inactive carriers characterized by persistent HBV infection of  liver without significant, ongoing necro-inflammatory disease. Eleven had liver cirrhosis characterized by clinical evidence (splenomegaly, ascitis) and paraclinical results, including low platelet count, prolongation of  prothrombin time and esophageal varices on upper gastrointestinal endoscopy. Liver cirrhosis was confirmed by liver biopsy. Patients were excluded if  they were co-infected with hepatitis C virus (HCV), hepatitis D virus (HDV) or human immunodeficiency virus (HIV).The following parameters were recorded for each patient from patient’s document in Tehran Hepatitis Center: sex, age, stage of  liver disease, alanine amino-transferase (ALT) level, aspartate transaminase (AST) and presence of  hepatitis B virus E antigen and anti-hepatitis B virus E antibodies. HBV genotypes were determined by using INNO-LiPA methodology (LiPA, INNO-LiPA HBV genotyping assay, Innogenetics N.V., Ghent, Belgium). The INNO-LiPA HBV genotyping assay is a line probe assay designated to identify hepatitis B virus genotypes A to G by detection of  type-specific sequences in the HBV-pol gene domain B to C. This method is based on the reverse hybridization principle. Biotinylated DNA material generated from the HBsAg open reading frame was hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane-based strips. After hybridization, unhybridized DNA was washed from the strip, alkaline phosphatase-labeled streptavidin was added and bounded to any biotinylated hybrid previously formed. Incubation with BCIP/NBT chromogen resulted in a purple/brown precipitate. Amplification of  appropriate the HBV genomic region was performed using the INNO-LiPA HBV DR amplification kit. The INNO-LiPA HBV genotyping strip contains 1 red marker line, 2 control lines, and 14 parallel probe lines. The conjugate control line is a control for the color development reaction and the amplification control line contains universal HBV probes to check for the presence of  amplified the HBV genomic material.www.wjgnet.comStatistical analysisData were analyzed with SPSS 11.5 software (SPSS Inc. Chicago, Illions, USA) using Student’s t test, χ2 test and Fisher’s exact test.RESULTSA total of  109 patients with a mean age of  37.17 ± 11.75 years, including 13% females and 87% males, were enrolled in this HBV genotype study. The distribution of  patients in different stages of  liver disease was as follows: 95 (86.4%) chronic hepatitis, 11 (10%) liver cirrhosis, and 3 (2.7%) inactive carrier. Of  the chronic hepatitis and liver cirrhosis patients, 26.4% were HBeAg-positive and 70% were HBeAg-negative. The mean serum ALT, AST, and ALP levels were 126.08 IU/L (88.46-163.71), 86.46 IU/L (49.54-123.39), 173.34 IU/L (152.74-193.94), respectively.Genotype D was the only detected type found in all patients. Mean age of  patients was significantly higher in the anti-HBe-positive group as compared with the HBe-Ag-positive group (P = 0.000). Also, the number of  the patients in the anti-HBe-positive group was significantly higher than the HBe-Ag-positive group (P = 0.019; Fisher’s exact test). Moreover, significant difference was found between the mean age of  patients with different stages of  liver disease. None of  the patients in the HBe-Ag-positive group had a normal ALT level. Most of  the patients in the chronic hepatitis stage had an abnormal ALT level in comparison with the liver cirrhosis stage (P = 0.024; Fisher’s exact test).DISCUSSIONClassifying HBV into genotypes has to be cost-effective and clinically relevant. It is imperative to collect more information on HBV genotypes from all over the world to reach a decision on their clinical utility[11].Data on the relation among the HBV genotypes, their pathogenicity in chronic liver disease including hepatocellular carcinoma and their effect on therapy are awaited with great interest, especially in Asia which is an endemic region of  blood-borne hepatitis viruses[15].Presently, based on an intergroup divergence of  8% or more in the complete nucleotide sequence, HBV can be classified into eight genotypes A-H, and different HBV genotypes are dominant in various parts of  the world[14,16]. The most important finding of  our study was that the only HBV genotype D was detected in all patients. The pattern of  distribution of  genotypes seemed to be simpler and was predominantly centralized into genotype D in all forms of  the chronic HBV infection. The results of  this study concur with previous studies, indicating that HBV genotype D prevails in the Mediterranean area, Near and Middle East, and South Asia[3,4,16]. For example, the result of  a similar study performed in Turkey showed all 44 patients studied had genotype D[16]. Another study in Yemen demonstrated that genotype D was the dominant genotype in a settled population, while genotype A was 5212      ISSN 1007-9327      CN 14-1219/ R       World J Gastroenterol      August 28, 2006    Volume 12     Number 32www.wjgnet.comfound only in communities with continuing African l inks [22]. In addit ion, one study in Egypt revealed genotype D was the most prevalent HBV genotype[23]. On the contrary, genotypes A, B and C were found to be predominant in Pakistan[24]. According to recent studies, genotype D in Asia is associated with more severe disease and may predict occurrence of  hepatocellular carcinoma in younger patients[9].After all, only less than 1 000 of  the 350 million persistent HBV infections have yet been genotyped. Continued efforts for understanding HBV genotypes through international co-operation will reveal further virological differences of  the genotypes and their clinical relevance.    REFERENCES 1 Magnius LO, Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 1995; 38: 24-342 Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schin-azi RF, Rossau R. A new genotype of hepatitis B virus: com-plete genome and phylogenetic relatedness. J Gen Virol 2000; 81: 67-74 3 Ogawa M, Hasegawa K, Naritomi T, Torii N, Hayashi N. Clinical features and viral sequences of various genotypes of hepatitis B virus compared among patients with acute hepati-tis B. Hepatol Res 2002; 23: 167-177 4 Ding X, Mizokami M, Ge X, Orito E, Iino S, Ueda R, Nakanishi M. Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases in Nan-ning, southern China. Hepatol Res 2002; 22: 37-445 Moriya T, Kuramoto IK, Yoshizawa H, Holland PV. Distribu-tion of hepatitis B virus genotypes among American blood donors determined with a PreS2 epitope enzyme-linked im-munosorbent assay kit. J Clin Microbiol 2002; 40: 877-8806 Mbayed VA, López JL, Telenta PF, Palacios G, Badía I, Ferro A, Galoppo C, Campos R. Distribution of hepatitis B virus geno-types in two different pediatric populations from Argentina. J Clin Microbiol 1998; 36: 3362-33657 Kao JH. Hepatitis B viral genotypes: clinical relevance and molecular characteristics. 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J Clin Microbiol 2003; 41: 1277-127918 Lin CL, Liao LY, Liu CJ, Chen PJ, Lai MY, Kao JH, Chen DS. Hepatitis B genotypes and precore/basal core promoter mutants in HBeAg-negative chronic hepatitis B. J Gastroenterol 2002; 37: 283-28719 Orito E, Ichida T, Sakugawa H, Sata M, Horiike N, Hino K, Okita K, Okanoue T, Iino S, Tanaka E, Suzuki K, Watanabe H, Hige S, Mizokami M. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 2001; 34: 590-59420 Adibi P, Ghassemian R, Alavian SM, Ranjbar M, Moham-madalizadeh AH, Nematizadeh F, Mamani M, Rezazadeh M, Keramat F, Ardalan A, Esmaeili A, Zali MR. Effectiveness of hepatitis B vaccination in children of chronic hepatitis B moth-ers. Saudi Med J 2004; 25: 1414-121821 Adibi P, Rezailashkajani M, Roshandel D, Behrouz N, Ansari S, Somi MH, Shahraz S, Zali MR. An economic analysis of pre-marriage prevention of hepatitis B transmission in Iran. BMC Infect Dis 2004; 4: 3122 Sallam TA, William Tong CY. African links and hepatitis B vi-rus genotypes in the Republic of Yemen. J Med Virol 2004; 73: 23-28  23 Saudy N, Sugauchi F, Tanaka Y, Suzuki S, Aal AA, Zaid MA, Agha S, Mizokami M. Genotypes and phylogenetic character-ization of hepatitis B and delta viruses in Egypt. J Med Virol 2003; 70: 529-53624 Idrees M, Khan S, Riazuddin S. Common genotypes of hepati-tis B virus. J Coll Physicians Surg Pak 2004; 14: 344-347 S- Editor  Wang J    L- Editor  Kumar M    E- Editor  Ma WHAlavian SM et al. HBV genotype in Iran                                                                        5213

Preliminary report of hepatitis B virus genotype prevalence in Iran

http://dx.doi.org/10.3748/wjg.v12.i32.5211

Preliminary report of hepatitis B virus genotype prevalence in Iran

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