Hepatitis in Blood Donors

The aims of the present study were to provide accurate prevalence of acute and occult hepatis B infection and hepatis C infection among potential blood donors in Cambodia and to study the accuracy of ELISA tests used for blood donor screening. A cross-sectional study was performed on samples collected from potential volunteer blood donors (n = 1,200) in two districts in rural Cambodia. The samples were tested using the ELISA technique for HBsAg, anti-HBc, and anti-HCV at a local blood bank. To validate the ELISA outcomes, a subset (n = 319) was analyzed by Automated Chemiluminescent Microparticle Immunoassay Technique (CMIA) at the University Hospital North Norway. The overall prevalence of the HBsAg positives was 7.7% (95% CI 6.2-9.3); the prevalence of anti-HBc positive samples was 58.6% (95% CI 55.8-61.4), and the prevalence of anti-HCV positive samples was 14.7% (95% CI 12.7-16.7). The prevalence rate of samples being both HBsAg positive and anti-HBc positive was 7.3% (95%CI 5.9 9.0), and the prevalence rate of HBsAg negative and anti-HBc positive samples was 51.2% (95%CI 48.4 54.1). The overall agreement between the ELISA and the CMIA test results was very high both for HBsAg and anti-HBc (kappa 0.93), and high for anti-HCV measurements (kappa 0.83). However, the false-negative rate for the ELISA anti-HCV test was as high as 15% (95%CI 6 30). endemic. Among transfusion transmitted infections, hepatitis B (HBV) is regarded as the most common, with risk estimates at 1:60,000 in countries where the prevalence is low. In areas where HBV infection is endemic, transmission rates are probably much higher, and infections occur in part due to improper testing (Wang et al, 2002; Hollinger, 2008). Blood donor screening for HBV surface antigen (HBs Ag) is carried out in low-income countries. However, HBV transmission may still occur during the iniINTRODUCTION Safe blood transfusions is a problem in developing countries where resources are limited and blood-transmitted diseases are SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 964 Vol 40 No. 5 September 2009 tial seronegative-window period of an acute infection, and also during late stages where the virus is still present (HBV-DNA positive) even though HBsAg is negative, so-called occult hepatitis B infection (OBI) (Liu et al, 2006; Bhattacharya et al, 2007). OBI may originate from recovered infections with persistent low-level viral replication, from escape mutants blocking export of antigen, or from reduced HBV replication after co-infection with HCV; HBsAg may or may not be present (Allain, 2004; Niederhauser et al, 2008). The infectivity of OBI is not clear, although several studies report the exclusion of anti-HBc positive donors regardless of an anti-HBs titer probably decreases the rate of HBV transmission by blood transfusion (Hennig et al, 2002; Behzad-Behbahani et al, 2006). One should take into account many studies of transmission risk may have methodological flaws that make it hard to interpret findings (Hollinger, 2008). Nevertheless, there are clear indications that both the viral load and the immune status of the recipient must be taken into consideration when assuming the risk for transmission of virus is higher in low-income countries, where large populations have deranged immune capacity from chronic malnutrition and endemic diseases. It is therefore urgent we develop scientific estimates of the infectivity of OBI in blood donations (Allain, 2007). In most Western countries the presence of antiHBc prohibits blood donation and thereby excludes the vast majority of potential OBI cases. However, due to limited resources and the potential exclusion of too many blood donors, this routine is seldom practiced in countries where HBV infections are endemic. Nucleic Acid Amplification (NAT) technology has enhanced accuracy in identification of OBI cases, without excluding blood donors who have previously been HBV infected but who are no longer carrying the virus. However, in low-income countries, especially in rural blood banks, NAT testing may not be financially or logistically feasible (Lieu et al, 2006). Studies from 15 years ago reported prevalence rates of HBV infection in Cambodia of 8% and HCV of 6.5% (Thüring et al, 1993). There are reasons to believe that access to modern medicine and vaccines have altered prevalences (Vong et al, 2005). However, to the best of our knowledge, no epidemiological studies of hepatitis B and C virus infections have been performed in Cambodia during the last decade. There seems to be large local variations in prevalence rates in Southeast Asia, with Vietnam alone reporting variations of HBV prevalence from 8% to 25%. Studies in Thailand have reported large prevalence variations among different populations (Ishida et al, 2002; Nguyen et al, 2007). Studies of prevalences in Southeast Asia have been done on relatively small study samples; consequently, the prevalence estimates are imprecise. The primary aim of this study was to provide accurate estimates of prevalence rates of acute and occult HBV and HCV infections among potential blood donors in rural Cambodia. The secondary aim was to study the accuracy of ELISA tests used for blood donor screening in Cambodian blood banks. MATERIALS AND METHODS This was a cross-sectional, epidemiological study with the reference population being potential blood donors in rural areas of Cambodia. The study was carried out in May and June, 2007 in the provinces of Battambang and Pailin, in the Kingdom of Cambodia. In order to detect differences in prevalences between the two communities HEPATITIS IN BLOOD DONORS IN CAMBODIA Vol 40 No. 5 September 2009 965 of at least 10% with a significance level of 95% and a test power of 90% given a baseline prevalence of HBV of 12%, a total of 1,200 samples were required for the study. The study unit was blood samples collected from 1,200 voluntary participants. Stratified sampling was applied to assess local prevalence variations; 600 samples were collected randomly in one remote area with a less developed infrastructure (Samlot), and 600 samples from one district which had a more developed infrastructure (Pailin, Fig 1). Prior to sampling, the potential participants and health care workers were informed that the study was related to setting up a safe blood transfusion service for the local population. They were also informed that the participation was voluntary and free of charge. The test results for each participant, with medical advice and individual counselling, were given back to them after testing at the Blood Transfusion Center in Battambang. Of the study participants, 677 were females and 523 males, the mean age was 32.8 years, with a range of 18 to 52 years. All participants were living permanently in the study areas and were not previously vaccinated against HBV. For validation of the local ELISA test, a subsample of 319 units was selected from the main sample and blindly re-analysed at the Department of Microbiology, University Hospital of North Norway. The subsample, 120 units for each test, was selected in order to detect test indicator differences of more than 5% with 95% confidence, assuming 2/3 being test-positive units and 1/3 being testnegative (Fig 2). There were no significant differences in the distribution of gender and age among the three subsets (Table 1). Blood collection and analysis The collection of samples took place at Fig 1–The two study areas in northwestern Cambodia. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 966 Vol 40 No. 5 September 2009 health centers and villages in the study areas. From each participant one blood sample of five ml was drawn into a sterile vacuum tube by a trained laboratory technician. The serum samples were set aside for spontaneous coagulation for 30 minutes before centrifugation. After centrifugation, serum was pipetted into two new tubes for further analysis. The serum samples were kept in a portable cooling box at 4oC and then taken to Battambang Blood Transfusion Center for analyses within three days. The main sample was analyzed using the ELISA technique (Monolisa® BioRad). The actual ELISA test has a claimed sensitivity and specificity for HBsAg of 100% and 99.94%; for anti-HBc of 99.53% and 99.5%; and for anti-HCV of 100% and 99.8%, respectively (Biswas et al, 2003). The subsample of 319 units was re-analyzed using an Automated Chemiluminescent Microparticle Immunoassay Technique (CMIA, Abbott, Wiesbaden, Germany). On CMIA testing of HBsAg, specimens with concentration values less than 0.05 IU/ml were considered negative and those that had values higher or equal to 0.05 IU/ml were considered positive. The CMIA analysis of anti-HBc and anti-HCV is based on the ratio of signal to cut-off value (S/CO). An S/ CO value less than 1.00 is classified as negative, and a value higher than 1.00 is classified as positive. Units with ratios in the range of 0.90-1.00 are classified as “equivocal” and were re-analysed twice (Murray et al, 2007; www.abbottdiagnostics).