In Pakistan, very little data is available about the IDUs reflecting an alarming situation of high infection rates among this highly vulnerable class of the society. Although most of these studies represent the data about HCV, for example, IDUs in Karachi have very high rates of hepatitis C (94%) which has also been documented in other reports as well [29, 30]. In few studies, HBV status has been assessed like; 7.5% IDUs were found to be positive for Hepatitis B surface antigen in Karachi reported by the very recent joint study conducted by Enhanced HIV/AIDS Control Program, Government of Sindh Pakistan and United Nations Office for Drug Control and Crime Prevention (UNODC), 2007. According to a survey regarding prevalence of viral hepatitis among the high risk groups like frequent blood recipients and IDUs is 13.05% while 25.67% patients of Chronic Liver disease harbor Hepatitis B virus . Inspection of 3 private health clinics in Hafizabad, Pakistan exposed disposable syringes and needles, used primarily for vitamin B complex, chloroquine, and penicillin, soaked in a bowl of tepid water. Extrapolation of study findings suggested 800 new cases of HCV and 109 HCV-related deaths in Hafizabad each year. Although this is referred to HCV but keeps equal importance as the risk of both HBV and HCV share equal importance using shared syringes. In another report, 350 IDUs were selected from a cohort study in Amsterdam of whom 70% injected recently, the prevalence of HIV, HBV and HCV were 31%, 68% and 65% respectively . Also, awareness about hepatitis B and hepatitis C as a result of sharing needles and syringes is less (60%) .
According to the quantitative data on injection usage and unsafe injection practices, such as the reuse of unsterilized syringe or needles, obtained by reviewing the published articles and unpublished reports of the WHO, 18 studies presented convincing evidence on the association of unsafe injection practices and the transmission of blood borne viruses such as hepatitis B and C, Ebola, Lassa virus infections and malaria . A simple mass-action model was applied to world census which showed that about 8–16 million HBV, 2.3–4.7 million HCV, and 80,000–160,000 HIV infections may result from unsafe injections each year .
Well established chains of transmission for blood-borne infections such as hepatitis and HIV exist in Pakistan. For example: needle sharing is common among the injecting drug users36. In 1999, UNDCP and UNAIDS Pakistan jointly studied the injecting drug users in Lahore, Pakistan's second largest city, finding an alarming high rate of hepatitis infection among them. Pakistan is a major transit and consumer country for opiates from neighboring Afghanistan, the world's largest producer of opium. As a result of the high levels of opium production in the region over the past two decades, Pakistan has now one of the highest addiction rates in the world . According to the statistics provided by Anti-Narcotic Force, out of 4 million drug addicts in Pakistan, 3% are women, 12% of whom inject the drug which lead to high risk of Hepatitis B/C and HIV/AIDS.
Estimations of a local NGO working for the well-being of addiction free population in Pakistan reports a total of about 4.1 million drug addicts, of which 2 million are chronic heroin addicts. Since the early 1980s, political and economic changes within the region have facilitated a dramatic increase of poverty and social problems linked to the illegal production and marketing of opiates. Pakistan itself has succeeded in supply reduction; nevertheless, the NWFP is seriously affected. The main factors contributing to the problem of drug addiction are: high level of illiteracy and lack of social and life skills; daily easy availability of a deadly substance with analgesic and relaxing properties considering it a potential source of relief for all kinds of stress. In Pakistan, 53% of heroin addicts start experimenting with drugs at the age of 15–25 years . NWFP has a striking figure of intravenous drug abusers locating in various regions of the province. Peshawar is located at the gateway of the transit trade route from Afghanistan easy availability of drugs at cheap prices are a permanent risk for Peshawar's youngster's and Pakistan's young population as a whole. Recent outbreak investigations of HBV in Sindh province, where the major etiological cause of the outbreak, was found to be the Intravenous drug injections among addicts signifies the IDUs as a major and potential transmission source of infection among the population.
In Pakistan, we have recently conducted a preliminary research project on prevalent genotypes of HBV in Pakistan (under publication) including general randomized population irrespective of major risk groups and have reported the prevalence of mixed genotypes infection in only 3% of total study subjects. However, here we have found that 28.57% IDUs are infected with multiple genotypes indicating very frequent horizontal transmission among the intravenous drug users.
The initial studies on HBV genotyping revealed that genotypes B and C are the most prevalent genotypes in Asian regions. It was because of the fact that all such studies were reported from Japan and China where genotype B and C are the most prevalent genotypes. Later on, it was found that all the seven HBV genotypes can be found in Asia . For instance, the predominant genotypes in India are Genotype A and D . The predominant HBV genotypes in Afghanistan were found to be genotype D . Similarly, Zeng reported 1.6% patients infected with multiple HBV genotypes .
Mixed infection with more than one HBV genotypes is of growing interest from the epidemiological, virological, clinical and therapeutic points of view. Research studies on such multiple infections are the vital requirement to understand the events of recombination that has been reported between different HBV genotypes [42, 43]. The multiple HBV genotypes determined in intravenous drug users (IDU) at Taiwan were as: mixed genotype A and B in 18 (5.5%); genotype B and C in 30 (9.2%); genotype B and D in 1 (0.3%); genotype A and C in 1 (0.3%); and mixed infections of genotype A, B, and C in 3 (0.9%) . In Belgium, the HBV genotyping studies revealed multiple genotype infection rate of genotype A and genotype D to be 8% and 9% for blood donors and gastroenterology patients . In Shenzhen (China), HBV infection rate with multiple genotypes was found to be 31% .
There are several studies reporting the existence of multiple HBV genotypes in various countries but mostly represent the figure related to patients irrespective of high risk groups like IDUs. For example, a study conducted on the patients from four regions of china reported that that mixed genotypes B and C was in 50.0% of the patients suggesting the mixed infection might lead to a severe damage of the liver tissue . 26% of liver transplant patients at London were reported to harbor multiple genotypes of HBV . In Tibet, the predominant HBV genotype is HBV C/D hybrid virus . Hannoun et al. (2002) found 8% of HBV patients with genotype mixture ; Toan et al., (2006) found that chronic patients are more prone to be infected with more than one HBV genotype than acutely infected patients ; genotypes mixture in HBV patients is also common in Thailand ; 16% HBV cases were positive for HBV genotype mixture in France .
We have found 28.57% patients infected with multiple (more than one) HBV genotypes. It has already been documented that the HBsAg prevalence rate in pregnant women was 2.5% in Pakistan, out of which 17% and 61% were HBeAg and anti-HBeAg positive thus indicating the vertical transmission a less important cause of HBV transmission . The present study reflects the importance of horizontal transmission as IDUs have several risky behaviors like sharing needles, cotton, syringes, multiple injections from a single drug source and jerking. The importance of such sources to identify remained the limitations of the study.