Distribution and clinical significance of hepatitis B virus genotypes in Pakistan

Hepatitis B virus (HBV) infection has been a major health problem as about 400 million people carry hepatitis B surface antigen worldwide [1], out of which, nearly 1 million die every year [2]. The infection rate of HBV has decreased significantly in developed countries like United States of America [3], where the acute HBV infection rate has fallen by 78 % during 1990–2005 [4]. Unfortunately, in developing and underdeveloped countries including Pakistan, the infection rate seems to have not decreased, even to any appreciable level. The failure in tackling the infection has been related to the high cost of antiviral drugs, lack of vaccination and public awareness about infection and the mismanagement to address the problem in Pakistan [5].

Eight HBV genotypes have been established on the basis of 8 % or more nucleotide divergence in the genome. These genotypes are named as A, B, C, D [6], E, F [7, 8], G [9] and H [10] which are common [11], while genotype I [12, 13] and J [14] are also introduced as new genotypes but their status is questionable. HBV genotypes have specific pattern of distribution in different geographic regions and ethnic groups of the world. Genotype A is prevalent in Brazil, USA, Canada, Northwest Europe, South Asia, Central African countries, Tunisia and Benin [15‐17]. Genotype B is common in Japan, Taiwan, Philippines, Hong Kong, China, Vietnam, Thailand, Indonesia and United States of America. Genotype C occurs in Australia, Polynesia, Melanesia, Micronesia, Indonesia, China, Hong Kong, Vietnam, Thailand, Japan, Korea, Taiwan, India, Solomon Islands, Brazil and USA. Genotype D is predominant in Mediterranean region, Spain, Albania, Czech Republic, Russia, Turkey, Middle East, Iran, Afghanistan, South Asia, Solomon Islands, Tunisia, Polynesia, Melanesia, Micronesia, Brazil and USA [17‐19]. Genotype E is endemic to Africa where it occurs only in some countries of the Western part of the continent while genotype F is widely distributed in new world. It has been reported from Alaska, Argentina, South America, Central America and Polynesia [17, 20]. Genotypes G has been recorded from North America, France and Germany, genotype H from Central America, South America and Mexico while genotypes I and J were reported only from Vietnam and Japan respectively [17].

HBV genotypes have been reported to have significant association with progression of liver diseases, risk of cirrhosis, development of hepatocellular carcinoma (HCC), viral load, HBsAg sero-clearance, HBeAg sero-conversion and response to antiviral therapy [21]. Co-infection with two different virus genotypes has been reported to be associated with worse prognosis of the disease. Some studies showed that co-infection with HBV/B and HBV/C is associated with high viral load and more severe liver disease as compared to single genotype infection [22, 23]. The patients with HBV/A have been reported to be more sensitive to interferon α treatment as compared to those infected with HBV/D [24] while the patients having infection with HBV/B have a higher response rate to interferon α treatment as compared to the patients infected with HBV/C [25]. Moreover, infection with genotype C alone was also found to be associated with significantly higher risks of liver cirrhosis and hepatocellular carcinoma as compared to genotype B infection [26, 27] while HBV/B shown an earlier HBsAg sero-clearance rate as compared to HBV/C [28]. In another study, HBV/A was found to have significantly higher sero-conversion rate of HBeAg as compared to HBV/B, HBV/C, HBV/D and HBV/F [29]. From all the studies mentioned above it’s quite clear that genotypes influence disease condition as well as response to treatment.

The picture regarding distribution of HBV genotypes in Pakistan and its association with disease stage has not been clearly understood yet. In fact, all of the available studies are mostly confined to specific cities/areas like Karachi (Sind) and Lahore (Punjab) and none of them contain data from other parts of the country. A vast range of areas from different regions of Pakistan still remains unexplored for HBV genotypes. Moreover, the existing state of knowledge regarding the distribution and prevalence of HBV genotypes in Pakistan seems to be sketchy and questionable too. For example, four studies [5, 30‐32] are in agreement that genotype D infection is most prevalent in the country while three studies [18, 33, 34] reported that genotype C is the most common ones in Pakistan. Yet in an another study, genotype C and D have been cited as most common HBV genotypes in Pakistan [35]. In view of the presence of the inadequate data, it was felt that a comprehensive study is required to determine the current distribution of HBV genotypes in all the six provinces of Pakistan and their possible association with different phases of HBV infection.

Out of total 840 samples, 125 were excluded from the study as HBV DNA was not detected in them. The remaining 715 samples were further analyzed (Table 1).

The present study is the first of its kind which included a cohort of 715 patients with chronic HBV infection from all six regions of Pakistan. The study analyzed the patients for HBV genotypes and their possible associations with liver disease severity in 384 consecutive chronic HBV carriers. According to the results, genotypes A, B, C, D, E and mix genotype infections with 6 different genotypic combinations are present in Pakistan, but the majority of patients (92.6 %) were infected with either genotype D, or genotype A or a mixture of both (A + D). The pre-dominant genotype in all regions was D followed by genotype A and mix genotype infection with A + D. Genotypes B, C, E and F were rare and collectively form only 1.5 % of the total prevalence.

HBV genotypes were not studied exclusively from all over the Pakistan so far. Table 4 summarizes some available data on the topic from Pakistan along with the results of present study. Most of the available data are from Karachi and Lahore or Punjab and Sind while none of the study has explored the regions of FATA, Gilgit-Baltistan, AJK, some parts of Khyber Pakhtoonkha province and most of the Baluchistan province. Though three studies carried out by Awan et al. [18], Alam et al. [30] and Idrees et al. [34] investigated the provinces of KP and Baluchistan additionally but the number of samples taken were not satisfactory to conclude the genotype distribution in these provinces (Table 4). None of the studies included the regions of Gilgit-Baltistan, AJK and FATA. Two of these three [18, 34] studies reported genotype C as predominant while one [30] found genotype D as predominant one. Genotype B which was not reported from Pakistan in any other study has also been reported in all three of these studies from 18 to 25 % of the samples. These results are contradictory to these studies.

All the studies confined to the samples from Punjab and Sind reported genotype D as predominant except one [33] which reported prevalence of genotype C and D as 75 and 25 % respectively. The remaining six studies [5, 31, 32, 37‐39] reported genotype D as the commonest one in Pakistan with percentage distribution of 59, 84, 70, 98.2, 84 and 93.2 % respectively (Table 4). The results of present study representing all parts of the country with significant number of samples is in agreement with the later group [5, 31, 32, 37‐39] that reported genotype D as the most prevalent HBV genotype in Pakistan. Genotype A was found to be the second more prevalent single genotype infection with 7.7 %. Genotype A was also reported in most of the previous studies [5, 18, 30, 32, 34, 39] in different proportions ranging from 0.8 to 21.4 % and a mean prevalence of 11.7 % (Table 4). Genotype B was present only in 0.6 % of present study samples while it was reported in proportions ranging from 18 to 24.5 % by previous studies from Pakistan [18, 30, 34]. None of the other studies reported genotype B infection, however mix infections involving genotype B were reported by some studies [18, 30, 31, 38] as high as 16 % of the samples (Table 4). Genotype C was detected in only 0.8 % of the samples in our study while three previous studies [18, 33, 34] have reported it as predominant genotype in Pakistan, whereas one study [39] reported it as second more prevalent genotype in the country. Genotype C was not reported by any of the other six studies (Table 4). Genotype E and F were not reported by any previous study except [18] which reported them in the proportions of 0.6 and 1.3 %. As already mentioned, genotypes E and F are endemic to Africa and the New World respectively [17, 20] and the fact that they were found in only 1 case (genotype E) was quite expected. Present study thus supports the conclusion that not only genotypes E and F but also genotypes B and C were rare in Pakistan (Table 4).

The relationship of HBV genotypes with liver disease is still not very clear. Although some studies have reported that HBV genotype influence the severity of liver disease and course of chronic HBV infection [26, 28, 40‐44]. Some other studies showed that HBV genotype had no influence on course of HBV infection [43, 45, 46]. Present study is in concordance with the later group [43, 45, 46]. Most of the studies compared genotype B and C in Asia and reported that the latter is more associated to severe liver disease than the former. On the other hand the genotype B is associated with an early HBeAg sero-conversion, more sustained remission after HBeAg sero-conversion, less active hepatic necroinflammation, a slower rate of progression to cirrhosis, and a lower rate of hepatocellular carcinoma (HCC) development as compared to genotype C [26, 28, 41, 43, 44]. A study from Pakistan compared genotype A, D and A + D for their association with complex liver diseases and concluded that genotype A is associated with more complex liver disease [47]. However, present study did not find any association of genotypes with disease stage. The relatively high prevalence of mix infection with genotypes A + D is a confirmation of the data reported by several previous findings about HBV genotyping in Pakistan [5, 18, 30, 32, 34, 37].

In conclusion, it has been observed in the present study that the distribution of HBV genotypes was almost similar in all the regions of Pakistan. Genotype D was the most common one (71.2 %) followed by mix infection with genotypes A and D (17.3 %) and genotype A (7.7 %). Although genotypes B, C, E and F were also detected but their prevalence was very low (1.5 %). Furthermore, genotype was not found to have any significant influence on liver disease progression in Pakistan.