The Role of Hepatitis B core Antibody in improving Blood Safety in Resource- Limited Countries Study on Voluntary blood donors Fayoum, Egypt

Background: Transmission of hepatitis B virus (HBV) via hepatitis B surface antigen (HBsAg) negative blood donors has been reported. HBsAg is still the only mandatory HBV screening test of blood donors in Egypt due to high cost of DNA testing of all collected blood. Many resource- limited countries have implemented screening antibodies to hepatitis B core antigen (anti-HBc) to further improve transfusion safety. The objective of study was to evaluate the significance of screening anti-HBc to reduce the risk of transfusion transmitted HBV infection in Egypt. 
Study Design and Methods: The study was conducted on 800 voluntary blood donors negative for HBsAg, hepatitis C antibody (HCVAb) and human immunodeficiency virus Ab. They were subjected to screening for anti- HBV core antibodies (total). Anti-HBc-positive samples were further tested for the antibodies to HBsAg (anti-HBs), and "anti-HBc alone" sera were tested for HBV DNA. 
Results: Among 800 healthy voluntary donors, 99 (12.37%) were anti-HBc-positive including 78 anti-HBs positive. The remaining 21 donors were anti-HBc alone, 2 of which (9.52%) were HBV DNA-positive. 
Conclusion: Greater consideration should be given to the implementation of anti-HBc as an additional screening test for blood donors in Egypt as the most cost-effective measure for further improvement of transfusion safety.


Introduction:
In Egypt, Current mortality due to liver disease, including liver cirrhosis and cancer, is over 40,000/year and is increasing annually. It is estimated that approximately 400 millionpeople worldwide are chronically infected with HBV,where Egypt is considered as an area of intermediateendemicity (29).Hepatitis B virus (HBV) is easily transmitted byblood products as both cellular and plasma-derivedcomponents may be infected (6) . Transfusion-transmittedHBV played a major role in the spread of this infectionsome decades ago all around the world, and is still athreat in developing countries, where the prevalenceof this infection is higher and the donor selection andscreening procedures are less tight (6,18) .
Occult hepatitis B infection (OBI) is defined by the presence of HBV DNA in the liver tissue of individuals who test negative for HBsAg, regardless of the detection of HBV DNA in serum (23) .
OBI was reported for the first time almost 30 years ago in a case report of HBV infection through blood transfusion by an antibody to hepatitis B core antigen (anti-HBc) only positive donor (33) .
In most developed countries,nucleic acid amplification testing (NAT) has beenintroduced along with serological testing for HBV, hepatitis C virusand human immunodeficiency virus in order to enhanceblood safety (8,24,1) .In contrast, developing countries with limited resources cannot implement NAT for screening of all donor collected bloodand are relying on proper donor selection which is complemented by sensitive cost effective serological screening tests to exclude the transmission of infective agents (19,21) .
In Egypt,screening for HBsAg is the only mandatory screening test for the detection of Hepatitis B virus (HBV) infection in blood banks (29) . Only four qualified blood centresare already implementing NAT in Egypt, and these are National Cancer Institute (NCI)blood bank, blood bank of Nasser Institute, blood bank of EL-Shabrwishy Hospital(Private hospital) and the EgyptianOrganization for biological products andvaccines (VACCERA) blood bank (27) . Given the constrained economy, lack of appropriate infrastructure and inadequate trained personnel limitits implementation in all blood banks (25,10) .New cost-effective strategies and stringent donor selection implementation are very important measures to ensure blood safety in resource-limited countries.
Unlike HCV, HBV cannot be cured, and the treatment for it is life-long.National Committee for the Control of Viral Hepatitis in Egypt estimated the cost of HBV treatment ranges from 250-550 Egyptian L.E/ patientmonthly according to the anti-viral treatment used, In addition to the expenses of advanced liver disease care (9) , it is clear that treatment costs are not comparable to the cost effective preventive measures that have to be implemented.
The aim of this study wasto determine the presence ofHBcAb and HBV DNA among HBsAg negative healthy blood donorsin Fayoum University Hospital Blood Bank to evaluate the significance of implementing screening anti-HBc to reduce the risk of transfusion transmitted HBV infection in Egyptian blood banks.

Blood samples
The present study included 800samples from blood donors negative for anti-hepatitis C antibody (HCV), anti-human immunodeficiency virus (HIV), and HBsAgcollected over a period of 6 months (from April 2014 to September 2014)inFayoum university hospital blood bank.

Detection of Serologic Markers
Serum HBV total anti-HBc was performed by ELISA technique,Monoliza Anti-hepatitis B core Plus-Bio-Rad, according to manufacturer's instructions on all donor samples.Positive samples were subjected to quantitative detectionof antibodies to hepatitis B surface (anti-HBs)with commercially available kits (ETI-AB-AUK-3, DiaSorin-Italy). Serum anti-HBs titers> 10 IU/L was considered positive.

Detection of Hepatitis B Viral DNA
HBV DNA level was estimated for blood units with low or undetectable serum anti-HBs levels"anti-HBc alone" using real time polymerase chain reaction (PCR) automated system.HBV

Serological results
All participants were negative for HBsAg. 99/800 (12.37 %) of blood donors were negative for HBsAg and positive for anti-HBc. Anti-HBs antibody was detected in 78/99(78.78%) of HBsAg negative and anti-HBc positive samples, with serum levels > 10 IU/L.

Molecular findings
Detection of HBV-DNA was performed on all samples that were negative for HBsAg and positive for anti-HBc antibody only by use of real time PCR technique. HBV-DNA was detected in 2 out of 21 anti-HBc positive specimens (9.52%).

Discussion:
OBI is defined as the presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals who tested negative for HBsAg (26) . Occult HBV is transmissible by blood transfusion, although the transmission rate is considered to be very low.
The clinical outcome of OBI transmission mainly depends on the immune status and copies of HBV DNA in blood products of the recipient (30) .At present, HBsAg detection is the only obligatory diagnostic screening test for HBV infection in blood transfusion centers in Egypt (25,10) . We examined 800HBsAg negative sera obtained from healthy blood donors and found that 12.37% of them were positive for anti-HBc, which is comparable to two previous Egyptian studies with a prevalence of 14.2% and 10.9 % of HBsAg negative volunteer blood donors (29,11) .The study is also comparable to the older anti-HBc prevalence rates reported among HBsAgnegative blood donors in India; 10.01% (19) and 11.2% in Syria (21) , respectively.
The prevalence of anti-HBc only in Europe and North America is overall quite low. A prevalence of 0.07% in the UK and 1.5% in Germany was reported (2,13) . In areas of higher HBV infection prevalence about 20%-70% of subjects are positive for anti-HBc antibody (7) .
In our study the overall prevalence of occult HBV infection in healthy blood donors was 9.52% among anti-HBc positive alone individuals. Different results have been reported in other studies regarding the rate of OBI in blood donors. These differences in the occult HBV prevalence may be attributed to race and ethnicity, geographical area and the HBV subtypes (3,14) . The frequency of HBV-DNA detected in HBsAg negative samples also variesconsiderably according to the prevalence of the infection. In Northern countries where the prevalence of chronic infection is less than 1%, no more than 5% of HBsAg negative /anti-HBc positive blood donor samples contain HBV-DNA (2,16) .In contrast, higher OBI levels in HBsAg-negative blood were recorded in several published reports. In India, the prevalence was 24% (22) and in a published study from Korea, 16% of the studied sample was found to be positive for OBI (15) .Other reports of theprevalence of HBV-DNA in only anti-HBc positive blood donors revealed 0% in Brazil (4) ,0.3% in china (28) , 1.1 % in Japan (31) , 3.2% in Saudi Arabia (5) and 12.7% in Ghana (32) .Some information is available regarding the infectivity of anti-HBc-only blood products or organs. The infectivity of blood donations containing anti-HBc as the only marker of HBV infection has been known for several decades and indicated that no more than 4% of recipients of anti-HBc-only blood developed HBV infection post-transfusion (17) However, Mosley reported 17% infectivity of antiHBc-only blood products (14,20) .Anti-HBc screening has the potential of excluding the vast majority of occult HBV infection but this exclusion of anti-HBc positive donors is impractical in countries where HBV infection is prevalent and higher than 20% of the populations are anti-HBc positive (12) . The use of HBV anti core testing to eliminate theresidual transfusion risk of transmission of HBV hasnot been evaluated in Egypt.

Conclusion:
One of the main mechanisms for OBI transmission is most likely through infected blood and its components and our findings revealed that OBI exists among Egyptian blood donors. Screening for HBsAg in blood banks in Egypt is not sufficient to completely exclude HBV infection in an intermediately endemic area like Egypt. NAT cannot be implemented for screening of all donor collected blood because of the high cost for a resource-limited country like Egypt. New screening policy to further increase the safety of blood transfusion is strongly indicated.
Anti-HBc antibody should be tested routinely on blood donor volunteers, and if the sera become positive regardless of anti-HBs titer, the blood should be discarded. Further testing for HBV-DNA is appropriate to follow up the blood donor patient for HBV infection.