Incidence of anti-HBc antibody ( IgG and IgM ) among HBsAg negative apparently healthy blood donors

In person who is unable to produce HBsAg, anti-HBc antibody is a helpful marker of hepatitis B virus (HBV) infection. In the present study, we have tried to find out the incidence of anti-HBc (IgG and IgM) among blood donors HBsAg negative. People came for donating blood voluntarily or for their relatives (n = 1000) was selected on inclusion and exclusion criteria. Purposefully selected and collated samples were first tested by HBsAg ELISA of third generation reagent. HBsAg negative sample was tested with anti-HBc ELISA. Positive found in the first test was retested. Out of 1,000 samples on duplicate test, 117 positives were detected. The incidence of antiHBc antibody among apparently healthy blood donors was found 11.7%. Article Info Depart e t of Tra sfusio Medi i e, Fa ulty of Medi i e, Ba ga a dhu Sheikh Muji Medi al U i ersity, Shah ag, Dhaka, Ba gladesh For Corresponden e: Nitya a da Shil a da.shil@g ail. o Re ei ed: O to er A epted: No e er A aila le O li e: De e er ISSN: O li e Pri t


Introduction
HBsAg is the first marker to appear in the blood, which persists, throughout the period of infectivity and eventually marks for chronic infection. 1,2In chronic carrier, HBsAg is eventually lost and replaced by anti-HBS.But within this, there is a period (diagnostic not detected by HBsAg window). 3But anti-HBc antibody is detected in all persons who are infected with Hepatitis B virus (HBV). 4In acute infection, high levels of IgM anti-HBc appear which persist for 3-4 months and are then replaced by IgG anti-HBc.High titer of IgG anti -HBc can be found in carriers.During the recovery phase of acute hepatitis B, anti-HBc may be present in the absence of HbsAg and anti-HBS. 5Anti-HBc may be the only circulating marker in such individual and they may only be identifiable by anti-HBc screening or by anti-HBc and HBsAg screening.In addition, anti-HBc screening having value in the detection of HBV infected donor who have mutant HbsAg not detectable by some HbsAg assay.In a small proportion of carriers, only anti-HBc can be detected in the plasma.Such subjects may transmit HBV by transfusion. 8 acute infection, high level of IgM anti-HBc have been found which persists for 3-4 months and then replaced by IgG anti-HBc.High titer of IgG anti-HBc can be found in carriers who may have low levels of IgM anti-HBc.So, mixed IgG and IgM assay is beneficial.It will detect both acute and chronic infection.
If anti-HBc can be detected in the plasma, such subjects may transmit HBV by transfusion.Subjects infected with HBV may be HBsAg negative due to point mutation in the precore region which may result in inability to synthesize HBsAg.Fulminant HBV infection developed in recipient of HBsAg negative blood from donors infected with this mutated virus.In all these donors, high levels of anti-HBc are present. 9out 50% of cases of HBV that could be transmitted by blood from HBsAg negative donors can be prevented by screening for anti-HBc. 4, anti-HBc screening, in addition to HBsAg, may help in reducing HBV transmission by transfusion.

Materials and Methods
This cross-sectional study was conducted from July 2015 to June 2016.One thousand cases of HBsAg ELISA negative samples of blood donors (properly selected on inclusion and exclusion criteria and properly screened for human immunodeficiency virus (HIV), hepatitis C virus (HCV), HBsAg, syphilis and malaria by rapid immunochromatographic tests) were included.

Abstract
In person who is unable to produce HBsAg, anti-HBc antibody is a helpful marker of hepatitis B virus (HBV) infection.In the present study, we have tried to find out the incidence of anti-HBc (IgG and IgM) among blood donors HBsAg negative.People came for donating blood voluntarily or for their relatives (n = 1000) was selected on inclusion and exclusion criteria.Purposefully selected and collated samples were first tested by HBsAg ELISA of third generation reagent.HBsAg negative sample was tested with anti-HBc ELISA.Positive found in the first test was retested.Out of 1,000 samples on duplicate test, 117 positives were detected.The incidence of anti-HBc antibody among apparently healthy blood donors was found 11.7%.Exclusion criteria were a) Under age 18 years and over 60 years, b) Weight less than 45 kg, c) cardiovascular diseases, d) History of surgery within one year with blood transfusion, e) History of blood transfusion for one year, f) Acupuncture, tattoo, ear or body piercing within 6 month and g) History of any known exposure to HBV and risk factor.

Article Info
The selected donors by interview with the help of a written questioner was then examined for physical fitness for donation was determined (Figure 1).These selected donor samples were collected in a 4'' test tube with a identification serial number 5 mL of whole blood collected and left for 24 hours at 4°C.Thereafter, the serum samples were separated and kept in Eppendrof tube and stored at below -20°C.When more than 90 samples were collected, they were tested for HBsAG-ELISA.For HBV, HIV, HCV, malaria and syphilis, departmental usual rapid screening process was followed.

ELISA testing for HBsAg
HBsAg was tested by ELISA method using commercial ELISA reagents of One step ELISA-HBsAg of Delta Biologicals, Italy of 3rd generation (sensitivity 100% and specifity 99.5%) Company's instruction was fully followed.HBsAg positive samples were disposed and new samples were included in the study with the same serial identification number.This is the final sample.Information about samples was noted in computer especially HBsAg and H/o jaundice.

ELISA testing for anti-HBC antibody
It was done with ELISA reagents of JAJ, USA.Samples positive were repeated.Repeated positives were taken as positive.Sensitivity of the reagent was not clearly cited in literature.Company's instructions were followed.

Results
Most of the donors (n = 963) belonged to age group of 18-30 years (Table I).Similarly, 91.5% were males.About 50% were students.
Out of 1000 samples on duplicate test 117 samples were found positive for anti-HBc (IgG and IgM).
The incidence was 11.7 percent.History of jaundice was found more among males (3.6%) than females (1.2%).
Anti-HBc antibody was found more among donors with the history of jaundice (3.3%) than that of females (0.3%).Donors having anti-HBc positive but no history of jaundice was among males 7.2% and among females 0.90 percent.

Discussion
Anti-HBc is found in all persons infected with HBV.Anti-HBc detects mainly HBV infection at diagnostic window period.It is only the HBV marker at the recovery phase of acute HBV infection.Anti-HBc is produced in the plasma shortly after HBsAg and remain in the circulation for 3-4 months.All noticeable risk factors were excluded from donors except history of jaundice, then anti-HBc was tested incidence found was 11.7%.Incidence was higher among aged donors than the young donors and the male than female.High incidence was among the illiterate person.World wide prevalence/incidence having different figure.In Pakistan it is 17.3%. 10In Spain, it is 4.8% among >65 years of donors.In literature, it is found that the isolated prevalence of anti-HBc is supposed to be 4-50% depending on sensitivity of reagent, age of donor.But the incidence of false positive is 11-3% found in 20 years prospective study.In North-West Greece, the incidence is 15.8%. 11In Italy identified mutant HB anti-HBc prevalence is 4.8. 11They proposed not to use mutant HB donor blood for transfusion.Now the question is whether can we propose to include anti-HBc as screening test in addition to HBsAg As, the reagent require repeated test, single test will loss huge number of valuable donors.There is no more record in our country about actual incidence/prevalence of anti-HBc.So, we cannot propose to include anti-HBc as screening of blood donor in addition to HBsAg.Now HB NAT is a alternative.But it require research about the new screening method.The cost of anti-HBc per test is not very high.But HBNAT is costly.But only HBsAg is not sufficient.We will have to decide which will be included or added in addition to HBsAg, HBsAg may be added to NAT or anti-HBc.In India, They are trying to include NAT with HBsAg.But NAT plus anti-HBc greatly raise blood safety 12 but of high cost.There is another benefit, about 50% of cases of HBV transmitted from HBsAg negative donors can be prevented by using anti-HBc testing.It is usually due to mutant HBV transfusion from HBsAg negative donor. 12A study about mutant HBV patient is essential.

Conclusion
The prevalence of anti-HBc Ab was high (11.7%) in Bangladeshi blood donors.

Nittyananda Shil, Jolly Biswas, Ayesha Khatun, Atiar Rahman, Nahid Sultana, Fatema Easmin and Sonia Sormin
Depart e t of Tra sfusio Medi i e, Fa ulty of Medi i e, Ba ga a dhu Sheikh Muji Medi al U i ersity, Shah ag, Dhaka, Ba gladesh A Jour al of Ba ga a dhu Sheikh Muji Medi al U i ersity, Dhaka, Ba gladesh Incidence of anti-HBc antibody (IgG and IgM) among HBsAg negative apparently healthy blood donors