Rajab Ali Khawaja  ( Department of Family & Community Medicine, King Khalid University Hospital, King Saud University Riyadh, Ministry of Health, Kingdom of Saudi Arabia. )
Asad Ali Khawaja  ( Paediatric Emergencies, Ministry of Health, Kingdom of Saudi Arabia. )

Hepatitis B virus is a crucial public health problem in Pakistan. It has been classified into eight genotypes (A-H) based on genome sequence divergence. Hepatitis B virus genotypes have distinct geographical distributions. Recently genotypes of hepatitis B have received a lot of attention due to its clinical as well as therapeutic importance. In this review, we will discuss the epidemiology, clinical outcome and response to antiviral therapy for different hepatitis B virus genotypes.

Hepatitis B virus infection is a global health problem with its continuously increasing morbidity and mortality in the developing countries like Pakistan. Despite the presence of hepatitis B vaccine, new Hepatitis B virus infection remains common. Worldwide, 350 - 400 million people are estimated to be persistently infected with hepatitis B virus and three-quarters of these people reside in Asia. According to WHO (World Health Organization), Pakistan falls under the endemic region with 3% HBV (hepatitis B virus) infected country population.^1-3

Long-term infection increases the risk of developing cirrhosis and hepatocellular carcinoma with approximately 1 million Hepatitis B virus-associated deaths from hepatocellular carcinoma every year.¹

Most acute infections with hepatitis B virus are self-limited with clearance of virus and development of immunity. However, an estimated 5% to 10% of adults and 85% to 95% of children develop chronic Hepatitis B virus infection.2 Studies indicated that the hepatitis B is a crucial public health problem in Pakistan with increased morbidity and mortality. Exposure rate of Hepatitis B virus in Pakistan is not known clearly but limited data shows 35-38% prevalence with 4% being carriers and 32% having anti-Hepatitis B virus surface antibodies through natural conversion.³

Currently hepatitis genotyping is emerging as a useful additional test that may add additional information and understanding of the natural history of hepatitis B virus. Recently a number of different Hepatitis B virus  genotypes have been discovered and received a lot of attention regarding their epidemiology, clinical implications and response to antiviral therapy. A problem in introduction of genotyping to clinical practice in the developing world is the lack of a simple, rapid and accurate test. Hence, despite the much progress in understanding the natural history of hepatitis B virus infection, we still have a long way to go before we can conquer hepatitis B virus infection.

Molecular Epidemiology of HBV Genotypes and its Geographic Distribution:

Hepatitis B virus is the smallest known DNA virus, and its partially double stranded circular genome consists of four overlapping genes encoding the viral envelop (pre-S and S), neocleocapsid (precore and core), polymerase with an error-prone reverse transcriptase activity, and X protein. Due to differences in the neocleotide homology of the surface gene there are different Hepatitis B surface antigen serotypes.⁴

Several methods have been developed and used to analyze hepatitis B virus genotyping which include, direct sequencing, PCR (Polymerase Chain Reaction) based restriction fragment length polymorphism (RFLP), line probe assay and enzyme-linked immunoassay. Recently a new genotyping method, based on PCR amplification assay using type-specific primers has been developed. Although the most common method for HBV genotyping is by PCR-RFLP technique, but it is reported that HBV genotyping by multiplex PCR is more sensitive than genotyping system using RFLP analysis.

Leblebicioglu reported that nested PCR methodology for HBV genotyping is 1000-fold more sensitive than PCR-RFLP.^5-9

At present, hepatitis B virus has been classified into 8 genotypes (A-H) based on intergroup divergence of 8% or more in the complete nucleotide sequence.^5-11

There is some evidence that the long-term prognosis, the initial clinical picture, and the response to treatment may differ depending on which genotype has infected the patient. Genotypes and sub-genotypes of Hepatitis B virus have different geographic distribution in the world (Table 1 and 2).^10,11 [(t1)] [(t2)] Prevalence of HBV Genotypes in Pakistan

Baig S, reported Genotype D as a dominant genotype followed by A and mixed A and D in Pakistan.¹²

In Karachi 180 chronic Hepatitis B surface antigen positive young females were tested for genotypes by Hakim ST et al3 revealed genotype D in 151 (84%) and mixed infection with B and D in 28 (16%). In a similar type of another study conducted by Masroor Alam et al, who tested 110 individuals positive for Hepatitis B virus infections indicated the highest prevalence of genotype D (65%), followed by genotype B (27%), A (5%) and co infection with multiple genotypes (3%).¹³

Another study was done in Pakistan to determine the prevalence of hepatitis B virus genotypes in patients with chronic hepatitis B infection. That study reported genotype C as the most dominant genotype in all of their cases, appearing in 41% patients. Further provincial breakup of the same study reported predominant prevalence of Genotype A (68%) in Sindh; genotype C (69%) in North West Frontier Province, whereas genotypes C and B were dominant in Punjab (40% and 26% respectively).¹⁴ Influence of HBV risk factors on HBV Genotypes:

A study conducted among injecting drug users in Pakistan showed presence of genotype D in 62%, genotype A in 9% while 29% individuals were found to be infected with a mixture of genotype A and D.¹⁵

Genotypes A and D were most prevalent in HIV-HBV co-infected patients in Madrid, Spain. HBV subtype A was present among three-fourth of patients infected through sexual contact (homosexual), whereas the same percentage of subtype D was isolated among injection drug users (P < 0.001).¹⁶ Influence of HBV Genotypes on Clinical Manifestation: Influence of HBV Genotypes on Antiviral Therapy: The response to antiviral therapy in hepatitis B virus infection is influenced by many host and viral factors. Recently, hepatitis B virus genotypes have attracted increasing attention since they influence the activity and outcome of Hepatitis B virus - associated chronic liver disease, as well as the response to antiviral therapies.

Lamivudine:

Since its approval in 1998, Lamivudine has gained wide popularity for the treatment of chronic hepatitis B due to high efficacy with minimal untoward effects.

Kao et al.²⁶ reported good response to Lamivudine in patients treated for 6-30 months infected with genotype B compared with C. Chien et al,²⁷ reported much higher sustained response to Lamivudine in patients infected with genotype B as compared with genotype C.

Other studies indicated that genotype D is more likely to have significantly higher sustained viral response (SVR) after Lamivudine therapy as compared to genotype A.²⁸ Another study based on 78 German patients reported that Lamivudine-resistant mutants emerged more rapidly with genotype A as compared to genotype D.²⁹

Interferon: Hepatitis B virus genotypes have been reported to correlate with response to interferon. Studies suggested that genotype B and A had a higher rate of Hepatitis Be antigen seroconversion compared to genotype C, D or E.^9,30-31

Peg-interferon a-2b is the best therapy to achieve Hepatitis B surface antigen clearance in patients with genotype A.32 Recent studies with pegylated interferon (IFN) confirmed that hepatitis Be antigen seroconversion occurred more often with genotypes A (47%) and B (44%) as compared to genotypes C (28%) and D (25 %).³³

A Erhardt et al reported higher sustained response (six months after treatment) to standard interferon alfa (IFN) therapy in hepatitis B virus genotype A compared with hepatitis B virus genotype D infected patients (49% Vs 26%) p < 0.005. He further  concluded that the sustained response to IFN for HBV genotype A compared with HBV genotype D was 46% Vs 24% (p < 0.03) in Hepatitis Be antigen positive (n=99) and 59% Vs 29% (p <0.05) in Hepatitis Be antigen negative (n=45) patients.³⁴ Thymosin alpha-1 therapy:

Genotype, presence of precore mutation and thymosin alpha-1 therapy were independently predictors to complete response. Genotype B compared to genotype C, is associated with a higher response rate to thymosine alpha-1 therapy.³⁵

Hepatitis B virus genotypes influence the severity of liver disease and response to interferon and lamivudine therapies.
Severity and outcome of chronic hepatitis B are more serious in patients infected with genotype C and A as compared with genotype B.
Evidence indicates a better response to interferon in patients infected with genotype A and B as compared with genotype C or D; whereas, the response to Lamivudin was better in patients infected with genotype B and D rather than genotype A or C.
Patients with Hepatitis B virus genotype A tended to be more likely to have lamivudine resistance mutations and to develop resistance earlier.

1. Lee W. M. Hepatitis B virus infection. New England Journal of Medicine, Dec 1997; 337: 1733 - 45.
2. Gust I, Crowe S. The global importance of viral hepatitis. Clin Trop Med Commun Dis 1986; 1: 281 - 301.
3. Hakim ST, Kazmi SU, Bagasra O. Seroprevalence of Hepatitis B and C Genotypes among Young Apparently Healthy Females of Karachi-Pakistan.  Received for publication on 04 September 2007. Accepted in revised form 11 October 2007. Libyan J Med. AOP: 071123. www.ljm.org.ly
4. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo R I, Imai M, Miyakawa Y, et al. Typing hepatitis B virus by homology in nuleotide sequence: Comparison of surface antigen subtypes. J Gen Virol 1988; 69: 2575 - 83
5. Kidd-Ljunggren K, Miyakawa Y and Kidd AH. Genetic variability in hepatitis B viruses. J. Gen. Virol 2002; 83: 1267 - 1280.
6. C L Lin, J D Chen, C J Liu. Clinico pathological differences between hepatitis B viral genotype B- and C-related resectable hepatocellular carcinoma. J Viral Hepatitis. 2007; 14: 64 - 69.
7. M Sugiyama, Y Tanaka, T Kato, E Orito, K Ito, SK  Acharya, et al. Influence of hepatitis B virus genotypes on the intra- and extra cellular expression of viral DNA and antigens. Hepatology 2006; 44: 915 - 24.
8. Yu MW, Yeh SH, Chen PJ, Liaw YF, Lin CL, Liu CJ, et al. Hepatitis B Virus Genotype and DNA Level and Hepatocellular Carcinoma: A Prospective Study in Men. J Natl Cancer Inst 2005; 97: 265 - 72
9. Zhang X, Zoulim F, Habersetzer F, Sidong X, Trepo C. Analysis of hepatitis B virus genotypes and pre-core region variability during interferon treatment of HBe antigen negative chronic hepatitis B. Journal of Medical Virology 1996; 48: 8 - 16.
10. Yuzo Miyakawa, Masashi Mizokami. Classifying hepatitis B virus genotypes. Intervirology 2003; 46: 329 - 38.
11. liu CJ, Kao JH, Chen DS. Therapeutic implications of hepatitis B virus genotypes. Liver international 2005; 25: 1097 - 1107.
12. Baig S, Siddiqui AA, Ahmed W, Qureshi H, Arif A. The association of complex liver disorders with HBV genotypes prevalent in Pakistan. Virol J. 2007; 4: 128
13. Alam MM, Zaidi SZ, Shaukat S, Naeem A, Sharif S, Mehar Angez, et al. Molecular epidemiology of Hepatitis B virus genotypes in Pakistan. BMC Infect Dis. 2007; 7: 115.
14. Idrees M, Khan S, Riazuddin S. Common genotypes of hepatitis B Virus. J Coll Physicians Surg Pak. 2004; 14: 344 - 7.
15. Alam MM,  Zaidi SZ, Shaukat S, Sharif S, Mehar Angez,  Naeem A, et al. Common Genotypes of Hepatitis B virus prevalent in Injecting drug abusers (addicts) of North West Frontier Province of Pakistan. Virol J 2007; 4: 63
16. Olmeda MP , Munez M, Samaniego JG, Rios P, Lahoz JG,   Soriano V. Distribution of HBV genotype in HIV patients with chronic HBV. Therapeutic implications. Aids research and human retroviruses 2003; 19 : 657 - 59.
17. 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: 19 - 22.
18. Chu CJ, Hussain M, Lok AS. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 2002; 122: 1756 - 62.
19. Sumi H, Yokosuka O, Seki N. Influence of hepatitis B virus genotypes on the progression of chronic hepatitis B liver disease. Hepatology. 2003; 37: 19 - 26
20. Kao JH, Chen PJ, Lai MY.  Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000; 118: 554 - 9.
21. Nakayoshi T, Maeshiro T, Nakasone H. Difference in prognosis between patients infected with hepatitis B virus with genotype B and those with genotype C in the Okinawa Islands: a prospective study. J Medical Virol. 2003; 70: 350 - 4.
22. Chu CJ, Keeffe EB, Han SH, Perrillo RP, Min AD, Soldevila-Pico C, et al. Hepatitis B virus genotypes in the United States: results of a nationwide study. Gastroenterology 2003; 125: 444 - 51.
23. Sugauchi Fuminaka. HBV genotypes and clinical manifestation among HB carrier in Thialand. J gastroentrol.hepatol 2002; 17: 671 - 76.
24. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterol 2001; 120: 1564 - 5.
25. Livingston SE, Simonetti JP, McMahon BJ, Bulkow LR, Hurlburt KJ, Homan CE, et al. Hepatitis B Virus Genotypes in Alaska Native People with Hepatocellular Carcinoma: Preponderance of Genotype F. Journal of Infectious Diseases 2007; 195: 5-11.
26. Kao JH, Liu CJ. Chen DS. Hepatitis B viral genotypes and lamivudine resistance. J Hepatol.  2002; 36: 303 - 4.
27. Chien RN, Yeh CT, Tsai SL, Chu CM, Liaw YF. Determinants for sustained HBeAg response to lamivudine therapy. Hepatology 2003; 38: 1267 - 73.
28. Thakur V, Sarin SK, Rehman S, Guptan RC, Kazim SN, Kumar S. Role of HBV genotype in predicting response to lamivudine therapy in patients with chronic hepatitis B. Indian J Gastroenterol 2005; 24: 12 - 5.
29. Zollner B, Peterson J, Puchmmer-Stuckle E, Kletzmayr J, Sternek M, Fisher I, et al. Viral features of lamivudine resistant hepatitis B genotypes A and D. Hepatology 2004; 39: 42 - 50.
30. Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes and the response to interferon therapy. J Hepatol 2000; 33: 998 - 1002. 
31. Wai CT, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with better response to interferon therapy in HBeAg-positive chronic hepatitis than genotype C. Hepatology 2002; 36: 1425 - 30.
32. Hajo J Flink, Monica van Zonneveld, Bettina E, Hansen, Robert A, de Man, et al. Treatment with Peg-Interferon a-2b for HBeAg-Positive Chronic Hepatitis B: HBsAg. Loss Is Associated with HBV Genotype. Am J Gastroenterol. 2006; 101: 297-303.
33. Janssen HLA, Senturk H, Zeuzem S, Akarka U, Cakalluglu Y, Simon K, et al. Peginterferon alfa 2b and lamivudine combination therapy compared with peginterferon alfa 2b for chronic HBeAg positive hepatitis B: randomized controlled trial in 307 patients. Hepatology 2003; 38: 1323.
34. A Erhardt, D Blondin, K Hauck, A Sagir, T kohnle, T Heintges, et al. Response to interferon alfa is HBV genotype dependent. Genotype A is more sensitive to interferon than genotype D. Gut 2005; 54: 1009 - 1013.
35. Y Tanaka and M Mizokami. Genetic diversity of HBV as an important clinical factor associated with differences in clinical outcomes. J infect dis. 2007; 195: 1-4.