In India screening for HBsAg by ELISA is at present the only mandatory test for detection of HBV infection in blood donations. Several reports around the world showed detection of HBV DNA in blood of HBsAg-negative individuals and suggested transmission of HBV infection by blood transfusion from HBsAg negative occult HBV carriers [26, 27]. Thus, evaluation of other HBV markers might reduce these risks.
Varying prevalence of antiHBc, a marker for exposure to HBV infection, has been reported from different parts of India; ranging between 8%-18% of total donor population. In the present study from Behrampur, Ganjam in Orissa, about 30.1% of total donations (220 of 729) was antiHBc positive indicating a very high rate of exposure to HBV infection among the blood donors from this region.
The Ganjam district in the state of Orissa in southeastern India records very high rate of migration in search of livelihood. Among the 3.1 million people living in 24 blocks in the district, people from 22 blocks depend on migration for their income . Here, the men remain away from home for eight to nine months at a stretch, in their sexually most active period . This also leads to a number of women left in Ganjam being involved in promiscuous habits . This unusual matrix puts migrants and their families at risk of sexually transmitted infections at various points . Notably around 50 per cent of the HIV positive people in Orissa are from Ganjam and most of them are migrant labourers and there is high prevalence of HIV among antenatal clinic attendees (3.25%), . Thus, the high rate of antiHBc prevalence, indicating exposure to HBV-a sexually transmitted infection, among the blood donors of Ganjam district is not unusual.
Prevalence of occult HBV infection was also high among antiHBc positive donations from the present study, 30% percent of antiHBc positive donations (66/220) being HBV DNA positive. Furthermore, among the 220 antiHBc positive donations 40 were antiHBs positive and are supposedly protected against HBV infection. Notably, 12/40 (30.0%) of antiHBs positive donations were HBV DNA positive, even one of them was with viral load ≥104 copies/ml. Among the 164 antiHBc only donors, 45 (27%) were HBV DNA positive (Table 1). The higher proportion of OBI found among the younger group (18-24 & 25-34) bears special significance as they are in the sexually active group and might possibly be related to the problem of frequent migration for livelihood. However, this requires further study.
The frequency of occult HBV infection varied considerably from different parts of the world according to the prevalence of HBV in the population. Prevalence of OBI is high in high HBV prevalence zone of the world and low in low HBV prevalence zone of the world. Studies from other parts of India reported occult HBV infection ranging from 21% in Kolkata (Eastern India), 20.87% in New Delhi (Northern India) to 0% in Chandigarh (Northwestern India) [7–9]. A study from Japan  reported DNA positivity of 38% in 19 of 50 anti-HBc reactive samples. While that from North America found 3.7%, HBV DNA positive among 107 anti-HBc positive/anti-HBs negative samples . Notably due to the low viral load in majority of these samples, detection of OBI required sensitive DNA amplification techniques.
There is increasing evidence that OBI is associated with chronic liver disease and HCC [31, 32], in addition to being a source of transmission of HBV by blood transfusion or orthotropic liver transplantation . Therefore, the high rate of OBI among blood donors of Ganjam district is of serious concern. As these donors have the potential to transmit HBV contaminated blood through the public blood supply even after donor screening for HBsAg. Thus, our data indicates OBI as an emerging infection hindering safety of blood transfusion in this community. Ideally, HBsAg negative individuals who are either antiHBc negative or those who are antiHBc positive/antiHBs positive/HBV DNA negative should be selected as regular blood donors to minimize transmission due to occult hepatitis B infection. This will help in the reduction of the transfusion associated transmission due to OBI; but will increase the testing costs.
Routine blood donor screening for antiHBc has been implemented in some countries resulting in a decrease in the risk of post-transfusion HBV infection . Hence, in our resource poor setting, inclusion of antiHBc testing for donor screening will definitely remove possible HBV infected donations. Thus though a large number of donations will be rejected, but rejection of these donations will be valuable in reducing the risk of HBV transmission with its potential consequences, particularly among immunocompromised recipients.
Phylogenetic analysis revealed that although HBV/D with subgenotype HBV/D2, HBV/D3 was predominant among OBI cases in this population, HBV/Cs, HBV/Aa and HBV/D1 were also present; this is in accordance with previous reports from other parts of India [20, 21, 34]. Interestingly, similar to eastern and northern India HBV/D3 was predominant among OBI cases while this subgenotype was mainly found among drug addicts of Europe . The most common amino acid substitution found within MHL region was T125 M (44/60), T118V (11/60) and A128V (11/60) found in HBV/D2 and HBV/D3. A stop codon was found in HBV/D2 at position 69th amino acid which results in a truncated HBsAg gene lacking the total 'a' determinant region, which might be a reason for HBsAg negativity making the surface gene nonfunctional. This mutation was previously documented in subgenotype D1 . Only one sample had M133T substitution in genotype HBV/D, previously described to be associated with diagnostic and vaccine failure . None of the other substitutions found were known to interfere in HBsAg assay. Consequently, a low HBV viral load seems the most likely explanation for our HBsAg negative/DNA positive samples. Some of these had substitutions that also caused amino acid mutations in the overlapping reverse transcriptase (RT) domain in the pol gene; therefore, the typically low level of HBV DNA observed in occult HBV infections, may be related to the mutation in RT domain which in turn resulted in low level of protein synthesis, as reported from eastern India .
In conclusion, our study underscores the high rate of exposure of HBV to the blood donors of Behrampur, Ganjam, a high HIV prevalent district of India. One in every 11 blood donors who are HBsAg negative/antiHBc-positive and one in every 3 antiHBc positive/antiHBs positive donors, have OBI, with possibility of transmission of HBV to transfusion recipients. Our results suggest inclusion of antiHBc testing for donor screening. Although addition of antiHBc testing, will lead to rejection of a large number of donor units; it will definitely eliminate HBV infected donations. This will help in reducing transfusion associated transmission due to OBI in this resource poor setting with its potential consequences, especially among the immunocompromised population. The significance of high rate of OBI should be monitored by further studies to evaluate the transmissibility and infectivity of OBI by blood transfusion. These data might lead to improvements in donor screening in the blood banks of India.