Hepatitis C virus (HCV) infection is one of the most important Flaviviridae infections with significant clinical problems throughout the world in humans and it is responsible for the second most common cause of viral hepatitis [1]. To date at least six major genotypes of HCV, each having multiple subtypes, have been identified worldwide [2]. The different genotypes are relevant to epidemiology, vaccine development, and clinical management of chronic HCV infection [3]. Furthermore, the HCV genotype is the strongest predictive parameter for sustained virological response [4]. This clinical relevance of HCV genotyping attracted attention from studies that reported an influence of HCV genotypes on the clinical course of disease and response to interferon therapy, as patients with different HCV genotypes respond differently to alpha interferon [5]. Firm evidence has been established that patients with type 2 and type 3 HCV infections are more likely to have a sustained response to therapy than patients with type 1 HCV infections [6]. Rates of sustained virological response to combination therapy in patients infected with HCV-2/3 and HCV-1 genotypes are 65% and 30%, respectively [7, 8]. Therefore the patient genotype should be taken into consideration when prescribing interferon standard therapy.

HCV genotypes 1, 2, and 3 appear to have a worldwide distribution and their relative prevalence varies from one geographic area to another. HCV subtypes 1a and 1b are the most common genotypes in the United States [4]. These subtypes also are predominant in Europe [9–11]. The predominant subtype reported from Japan is subtype 1b that is responsible for up to 73% of cases of HCV infection [12]. HCV subtypes 2a and 2b are relatively common in North America, Europe, and Japan and subtype 2c is found commonly in northern Italy. HCV genotype 4 appears to be prevalent in North Africa and the Middle East [13, 14], and genotypes 5 and 6 seem to be confined to South Africa and Hong Kong, respectively [15, 16]. HCV genotypes 7, 8, and 9 have been identified only in Vietnamese patients [17], and genotypes 10 and 11 were identified in patients from Indonesia [18]. There has been disagreement about the number of genotypes into which HCV isolates should be classified. Investigators have proposed that genotypes 7 through 11 should be regarded as variants of the same group and classified as a single genotype, type 6 [19, 20].

From Pakistan few studies are available on the distribution of various hepatitis C virus genotypes based on small sample sizes [13, 21, 22]. No study is available on geographic variation in the prevalence of various HCV genotypes and various routes of transmission for different genotypes from Pakistan. Therefore, this study was initiated to determine the frequency distribution of various HCV genotypes and subtypes present in different geographical regions of Pakistan and to determine various risk factors for its transmission.

Data so obtained from different parts of the world have focused on the increasing interest of HCV genotyping by mass screening as it is useful for the solution of epidemiological questions and development of vaccines against HCV. Furthermore it has been shown to be beneficial to facilitate therapeutic decisions and strategies [25–27]. It has been demonstrated that the severity of the disease, its progression and response to therapy may vary according to the genotype [10, 28].

Substantial regional differences appear to exist in the distribution of HCV genotypes. Thus knowledge on the distribution of various genotypes in our country is essential for its prognostic implications in chronic hepatitis C infection. In the present study the frequency of various genotypes of HCV present in Pakistan and various risks for possible transmission were observed. All serum samples tested were HCV-RNA positive by PCR and could thus be genotyped. However, genotype-specific PCR products were seen in about 94% serum samples by this molecular biology-based system that is an excellent percent of the sample size. The data of the present study correspond well to previous studies, in which genotypes or serotypes were determined from other regions of the world [29–31]. The presence of various HCV genotypes such as 1a, 1b, 2a, 3a, and 3b reported in this study have also been reported in the earlier studies with predominated genotype 3 in Pakistan [21, 22]. Though these studies were done with lower sample sizes, the reported genotypes frequencies are the same as observed in the present study. Additionally six more genotypes such as 1c, 2c, 3c, 4, 5a and 6a were isolated for the first time ever from this country in the present study. The frequency of genotype distribution in the present study also seems to be quite similar to that reported from other South Asian countries such as Nepal [17] and India [32, 33] where genotype 3 was found as the predominant genotype but different from those in Japan [34], Thailand [35], Vietnam, USA and Western Europe [36] where genotype 1 is the most prevalent HCV genotype.

When the results of the present study on HCV genotypes were compared to the genotyping results obtained in other studies from Pakistan [21, 22], it was observed that the frequency of genotype 1 is increasing in this country without any increase in the frequency of genotype 3. It seems that in coming 15–20 years the current most prevalent genotype 3a will be replaced by less common genotype 1 (a or b). If this actually happens here in Pakistan, it will complicate more the present situation of HCV that is a big health problem in this country. Recently replacement of one HCV genotype (type 1b) by another (type 2) has been reported from Venezuela that took only 10 years time for this displacement [36]. We also suggest confirming this observation by conducting other studies on HCV genotypes. Another interesting finding of the study is the isolation of genotype 4 for the first time from Pakistan although this genotype is believed to be absent from Pakistan [22]. HCV genotype 4 is prevalent in the Middle East and Central Africa; however, our patients did not have a history of visit to these areas of the world. Genotype 4 has been reported to be frequently associated with cirrhosis and a poor response to interferon [37, 38]. Like Pakistan, HCV genotype 4 is also rare in the United Sates and there are few published data regarding response to therapy in patients with HCV genotype 4 infections in Pakistan and United States [39]. None of the genotype 4 having patient has history of visit to Middle-East.

In order to discern if there are any regional differences in HCV genotype distribution in Pakistan, the distribution for this population was examine by individual Provinces. No regional difference with respect to HCV genotype distribution was seen in the Provinces of Punjab, N.W.F.P and Sindh where the most prevalent genotype is 3a followed by 3b. But a clear difference was observed in the Province of Balochistan where the predominant genotype found was 1a followed by 2a and 3a. Balochistan shares a long boarder with Iran in the west where genotype 1 is the predominant genotype in the region [40]. It is quite possible that genotype 1 may enter to Pakistan from Iran through local persons who cross boarder for jobs and trade. HCV genotypes distribution described in this study does not present modification by gender, and the distribution of HCV genotypes was similar both in male and female HCV patients with no significant difference seen for various genotypes in male and female patients (P < 0.05). The results of the present study indicate that HCV genotype distribution varies with age. It has already been reported that HCV genotype distribution varies with age in both male and female patients [41]. Subtypes 1a and 1b were found most prevalent among older patients, whereas subtype 2a/2b and 3a/3b were mainly found among younger ones.

Some studies suggest that different types of HCV may be associated with different transmission routes. In the present study majority of the patients seem to have more than one route of transmission however we took the most probable one for data analysis. In Pakistan genotype 3a is the most prevalent genotype and more than 86% patients with genotype 3a received multiple injections. It is quite possible that this type was spread in Pakistan by doctors, vaccination teams and other medical persons using non-disposable syringe before 1990 when one syringe was used for injections to all attended patients. This type of practice is still there in the rural areas of the country. This observation is supported by a study where subtype 3a appeared to be prevalent among injection drug users and it is believed that it was introduced into North America and the United Kingdom with the widespread use of heroin in the 1960s [42]. Further, it has also been reported that HCV genotype 3a is particularly prevalent in intravenous drug abusers in Europe and the United States [43]. For genotype 1a and 1b, the probable causes of transmission observed in the present study were major/minor surgeries and dental procedures. This is in agreement with a study from Hungry where majority of the cases with genotype 1 has a history of hospitalization for major/minor surgery, dental procedures and shaving by barbers. In contrast in one study, 95% of all patients with genotype 1 were infected with subtype 1b and majority of those patients had received transfusions of blood and/or blood products [30]. Genotype 2a was more frequently seen (>50%) in case with history of no apparent causes of transmission. Mode of transmission was not clear for more than half of patients with untypable genotypes. Type 4 is uncommon in this country and its route of transmissions were surgeries (38%) followed by reuse of syringes (22%). As expected, the prevalence of HCV mixed-genotype infections was high (52.79%) in thalassaemic patients who had received multiple blood transfusions.

It appears that more than 70% of the cases were acquired in hospitals or other medical facilities because a large number of subjects (61.45%) received therapeutic injections and surgeries (10.62%) from the clinics of local general practitioners in both government and private hospitals. It has already been reported that Pakistan has one of the highest frequencies of injections in the world and the average number of injections per person per year is more than 9 injections (44–45). Further been reported in the same studies that 49% of patients receive injections at their first outpatient visit where in addition to the unnecessary use of injections, safe injection practices are not followed. Furthermore, previous vaccination have also been alleged to ignore safe injection practices that was largely received at the public health-care facilities including the use of used syringes [44, 45]. As shown in the present study and also reported in previous other studies [44, 45] subjects who received more injections were more likely to be infected with HCV and these non-sterile syringes or needles may be the source of HCV infections. According to a survey conducted by Ministry of Health, in Pakistan more than 72% therapeutic injections and 50% immunization injections in public health-care facilities are unsafe and potentially dangerous [46]. In these studies it is further shown that the use of multiple-dose vials is common in many government and private sector hospitals of Pakistan that is the major risk factor for the transmission of HCV infection [44–46] especially genotype 3a.

The predominant HCV genotype in Pakistan is type 3a followed by 3b and 1a. Six HCV genotypes such as 1c, 2c and 3c, 4, 5a and 6a were isolated for the first time from Pakistan during the study. The frequency of genotype 1 was observed to be increasing in this country without any increase in the frequency of genotype 3 that may be very dangerous in coming 15–20 years. Regional difference in genotypes was observed only in Balochistan province of Pakistan. More than 70% of the cases in Pakistan are hospitals acquired.