SITUATION OF HEPATITIS IN PAKISTAN AND PREPARATION OF DIAGNOSTIC REAGENTS FOR THE DETECTION OF HBsAG (ELISA)

Hepatitis is inflammation of liver that may be caused by different viruses, bacteria, drugs, or excessive alcohol intake. The most common causative viruses are hepatitis A, B, C, D, E, F and G. Hepatitis A and B viruses were identified before 1970. In 1977 delta agent was identified. It occurs as a coinfection or superinfection in persons who have suffered from hepatitis B recently or in the past. Non A--Non B hepatitis has been identified as hepatitis C virus and hepatitis E virus. Hepatitis C includes most of the transfusion associated hepatitis. An epidemic form of hepatitis in the Asian continent has been designated as Hepatitis E virus (HEV). All forms of viral hepatitis pose serious public health problems world wide. In the United States viral hepatitis ranks second among the reportable communicable diseases. In our country the prevalence rate is very high and it falls in the intermediate to high endemicity zone of hepatitis prevalence. Hepatitis A has a world wide incidence. The prevalence decreases with increasing age and increases with low socio-economic status. Hepatitis B is endemic in Africa, Eastern Europe, the Mediterranean, Asia, and South America. In these areas more than 50 percent of the population have been exposed to Hepatitis B virus. Delta hepatitis is also found in areas of high hepatitis B prevalence. Hepatitis C has a prevalence rate of 0.2 to 1.5 percent in random blood donors in the United States. Europe and Japan have a high rate of about 80 percent in high risk groups such as intravenous drug abusers. Hepatitis E is endemic in areas of overcrowding, unsafe water supplies and inadequate disposal of human waste. It is found as acute disease in young adults. Hepatitis A and E are transmitted via oral fecal (enteric) route. Hepatitis B, D and C are transmitted via parenteral route through exposure to contaminated blood, primarily through transfusion of infected blood or the sharing of infected needles. Sixty to eighty percent of babies born to HBV infected mothers become carriers of infection and spread the virus for the rest of their lives. Classic symptoms of hepatitis include jaundice, fever and abdominal pain. Many patients have flue like symptoms. The viruses can only be differentiated through specific serologic testing.\ud \ud Hepatitis is one of the 10 reportable diseases in Pakistan. Hepatitis A is a childhood disease. In cases of hepatitis under ten 60 percent of the cases are due to Hepatitis A, 30 percent HEV, 10 percent HBV. About 70 percent of the children under ten have IgG antibodies against hepatitis A and they last longer and protect them from hepatitis A in future. Seventy percent of the all new born have transplacental IgG antibodies against hepatitis A that last about 8 months of age. In adults more than 66 percent of our population is immune to hepatitis A. Ninety five percent of the. blood donors had IgG antibodies against hepatitis A. It is equally prevalent in male and female and there is no significant difference in rural and urban populations.\ud \ud Pakistan is endemic area for viral hepatitis B. It is more aggressive due to co-infection and super-infection with delta virus. The case fatality rate with delta infection is 30.2 percent in Sind and Punjab. Intra-familial spread of hepatitis B is quite high. The highest reported in spouses is 23.5 percent. Pakistan has a high carrier rate of hepatitis B. It is between 10 to 14 percent with ELISA and RIA techniques respectively. Hepatitis B is more prevalent in males as compared to females (8.13% / 6.7% respectively). Transplacental transmission from mother to infant is not the major route of transmission for HBV, as none of the infants of HBV positive mothers had HBsAg in cord blood. The route of transmission of hepatitis B in infants is horizontal from mother to infants as they are close to mothers during infancy. Hence infants of hepatitis B positive mothers must be vaccinated against hepatitis B. Sub types of hepatitis B have different geographical distribution. The four major sub types of HBsAg are ayw, adw, adr, and ayr. In our country, the most prevalent sub type is ayw (78-95%), adw prevalence is (2.4-14%), adr (1.0-7.7%) and ayr (2.4%). Delta hepatitis poses a common community health problem in Pakistan.\ud \ud In patients with chronic and fulminant type of hepatitis 52 percent of the cases among hepatitis B patients had delta infection. The death rate in cases with delta infection was 30 percent and it was associated with fulminant type of hepatitis. It was also studied in dialysis patients and the incidence of delta infection was 14 percent in patients who had hepatitis B during dialysis. The persons who have recovered after hepatitis B infection develop antibodies against hepatitis B. More than 17.6 percent of the random blood donors were found positive for anti-HBs. Immune status of medical and para medical staff was 45 percent as compared to 28 percent in matched controls. In a number of studies the prevalence of anti-HBs was 12.2 - 40 percent.\ud \ud Non A Non B is now identified as hepatitis C and E virus infection. Its incubation period is 6 to 8 weeks. It causes a milder form of acute hepatitis than Hepatitis B but 50 percent individuals develop chronic infection following exposures. It may cause chronic liver disease and hepatocellular carcinoma. It spreads through blood products, blood transfusion, organ donation, and intravenous drugs abusers. Hepatitis C may be community acquired but the mechanism is not clear. It is endemic world wide and has high incidence in Japan, Italy, Spain and South Africa. In Pakistan at least four percent of the blood donors have HCV antibodies. Hepatitis E is identified as the cause of enterically transmitted water borne epidemics. HEV epidemics have been reported from different parts of our country. The cause was contaminated water supply. HEV is becoming a threat because of increasing population and the rusting of the water supply pipes. The largest out break of Hepatitis E was reported in 1993-1994 in the capital city of Islamabad. It affected 3827 people and was due to contamination of water supply with sewage. Nearby areas with independent and clean water supply were spared.\ud \ud Most of the laboratories in Pakistan have all the necessary facilities for conducting research on hepatitis. These are comparable to the laboratories in the developed countries. Pakistan uses reagents and kits in the Public Health Pathology laboratories that are imported and we have not been able to produce the immunological and biochemical reagents locally. The preparation of Immunological reagents is a tedious and time consuming process. locally produced anti-sera by our own purified strains would be more specific and would produce 'more sensitive reagents for diagnostic purposes. Imported kits nowadays are very expensive; for example an ELlSA kit for the detection of HBsAg from ABBOTT laboratories at present costs Rs. 18000/- for 100 tests. The cost/test comes to at least Rs. 250/-. In comparison our own produced standard ELlSA would cost about 1/10 of the price of the imported kit. This was one of the objectives that was kept in mind to develop ELlSA for detection of HBsAg. The reagents produced were compiled in the form of kit with all the necessary buffers needed for the test. The kits were sent for evaluation to the local laboratories and the evaluation reports were encouraging. The reagents were improved in the light of feed back from the laboratories. It can be said with confidence that the reagents are of standard quality and are produced by using local sub-type specific strains of HBsAg that was purified from our carrier blood donors. The purified antigen is used to develop the antibodies by hyper immunization of goats and rabbits. Purified anti-sera (lgG anti HBs) is used (rabbit anti-HBs) for coating the polystyrene plates as solid phase. The purified goat anti-HBs was conjugated with horse radish peroxidase to make conjugate. The substrate used was orthophenyalline diamine (OPD) that developed a yellow colour with HRP and was read at O.D. 492. This is the first preparation of the immunological reagents for the detection of HBsAg by enzyme immunoassay which may be the beginning of development of expertise for the production of other immunological reagents and related material