Original Article

INTRODUCTION

It is the prime responsibility of a Blood TransfusionService (BTS) facility to offer its patients the safest possibleblood for transfusion that is free from various TransfusionTransmissible Infections (TTI’s). Although measures suchas adoption of strict donor selection criteria, encouragementand maintenance voluntary non remunerative pool of blooddonors and temporary or permanent deferral of those withhigh risk behaviors judged by the use of questionnaire are aroutine practice globally, the final decision on whether ornot to use a blood/blood product for transfusion relies on theresults of infectious marker tests. In India, it is mandatory totest for HIV, HBsAg, anti HCV, Malarial Antigen andsyphilis. The use of sensitive screening methods that maydetect infectious agents during the ‘window period’ such asnucleic acid testing further adds to blood safety.

Hepatitis B virus (HBV) and hepatitis C virus (HCV)infections account for a bulk of acute and chronic liverdiseases world-wide [1-2]. Since, both the viruses sharesimilar risk factors and modes of transmission, a combinedHBV and HCV infection is frequently encounteredespecially in the HBV endemic areas. It has been shown thata dual HBV and HCV infection is associated with a moresevere liver disease, and is at an increased risk forprogression to hepatocellular carcinoma (HCC) [3].

Individual literature on the prevalence of HCV as wellas the Hepatitis B core antibody in different populationsworld over is readily available, however not many havestudied the two in conjunction with each other. Presence of

298 Apollo Medicine, Vol. 7, No. 4, December 2010

PREVALENCE OF ANTI-HCV ANTIBODIES AMONG HEALTHY ASYMPTOMATIC INDIANBLOOD DONORS AND THE CURRENT ROLE OF ANTI-HBC SCREENING AS A

SURROGATE MARKER FOR HCV INFECTION.

RN Makroo#, Aakanksha Bhatia*, NL Rosamma** and Minimol***#Director,*Registrar, **Senior Technologist, ***Technologist, Department of Transfusion Medicine,

Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.

Correspondence to: Dr RN Makroo, Director, Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections account for a bulk of acute and chronic liverdiseases world-wide. Since, both the viruses share similar risk factors and modes of transmission, a combinedHBV and HCV infection is frequently encountered especially in the HBV endemic areas. Until lately anti-HBcantibodies were considered as surrogate marker for HCV infection. But with the development of advancedtests for HCV detection the role of anti-HBc in this regard stands uncertain.

Key words: Combined HBV and HCV infection, Anti HBc antibodies, Surrogate marker.

anti-HCV and anti-HBc antibodies in absence ofdetectable HBsAg by ELISA has been reported in blooddonors in variable frequencies. Although blood units insuch cases are definitely discarded, these cases create adilemma where donor notification is concerned. Untillately anti-HBc antibodies were considered as surrogatemarker for HCV infection. But with the development ofadvanced tests for HCV detection the role of anti-HBc inthis regard stands uncertain [4].

Patients with HCV-related chronic liver disease (CLD)frequently show markers of previous HBV infection.Moreover, they may carry occult HBV infection. It hasbeen shown that co-infection with hepatitis delta virus orHCV results in down regulation of HBV replication and areduction in antigen synthesis [5]. It was initially believedthat the HCV core protein inhibits HBV replication andgene expression.[6] Bellecave, et al. [7] on the contraryshowed that HBV and HCV can replicate in the same cellline without in vitro interference. Thereafter other indirectmechanisms of viral interference mediated by innate and/or adaptive host immune responses were proposed [8,9].

MATERIALS AND METHODS

All donors who donated blood at the IndraprasthaMedical Corporation Limited Blood Bank, IndraprasthaApollo Hospital, New Delhi from January 2006 toDecember 2010 were enrolled in the study. The donorswere apparently healthy adults between the ages of 18-60years. All donors were subjected to a pre test counselingwhich was done by qualified staff trained to screen donors

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299 Apollo Medicine, Vol. 7, No. 4, December 2010

for blood donation as per the Drug and Cosmetics act.Donors who did not fulfill the general criteria for blooddonation, paid and commercial donors and those with ahistory of high risk behavior were deferred from donatingblood at our institution and hence, excluded from the study.

Post donation all donor blood samples were tested forthe presence of anti-HBc antibodies using Enzyme LinkedImmuno Sorbent Assay (ELISA). Third generation ELISAkits (MonolisaTM Anti-HBc PLUS, BIO-RAD) using fullyautomated EVOLIS walk away system (BIO-RAD) wereused for testing. For anti-HCV antibodies, third generationELISA kits (MUREX ANTI-HCV (VERSION 4) MUREXBIOTECH.) were used from January 2006 till October2010. From October 2010 till December 2010 SP-NANBASE C-96 3.0 kits from GENERALBIOLOGICALS CORP. were introduced. All samples thattested positive by ELISA were repeat tested in duplicateusing the same ELISA kit. Only repeat reactive sampleswere labeled as ‘POSITIVE’.

Nucleic acid testing (NAT) was done for all donorsamples using the TMA (Transcription MediatedAmplification). The Procleix Ultrio Assay, a qualitative invitro nucleic acid amplification test from Chiron(Novartis), for the detection of human immunodeficiencyvirus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA,and hepatitis B virus (HBV) DNA was performed and TheProcleix HIV-1, HCV, and HBV Discriminatory Assayswere run for the reactive cases. ELISA and NAT resultswere compiled, tabulated and analyzed.

RESULTS

A total of 99,131 donors were included in the study andtested for anti-HCV and anti-HBc antibodies by ELISA.The year wise distribution of blood donors and markerpositivity is shown in Table1.

From 2006 till 2010, anti-HCV antibodies weredetected in 406 donors, with an average of 81 cases peryear. These constitute an average of 0.41% of the totaldonors tested. Anti-HBc antibody on the other hand wasdetected in 10,169 donors (average of 10.25%) irrespectiveof the HBsAg status. Ninety Six (0.09%) showed positivityfor both anti-HCV and anti-HBc antibodies. In three out ofthese 96 donors HBsAg was detected indicating a clear cutcase of dual infection of HBV and HCV. However, in theremaining 93 donors only anti-HCV and anti-HBcantibodies were detected (Fig.1).

All the 96 cases were subjected to NAT. The initialProcleix Ultrio assay was reactive in 44 cases.Discriminatory assays confirmed the presence of HCVRNA in 42 out of these 44 cases, while the remaining twowere diagnosed having HBV DNA. Retrospective analysisrevealed that in one of these two cases HBsAg was detectedin ELISA, while the other one was only positive for antiHBc antibody besides Anti HCV.

A retrospective evaluation of the NAT positive casesalso revealed that there were merely 4 cases in the five yearstudy period where anti HBc positivity in absence of antiHCV yielded HCV RNA in NAT. Further in 3 out of thesefour ELISA also showed HBsAg positivity.

DISCUSSION

Hepatitis C is a leading cause of transfusion relatedhepatits, the transmission primarily being parenteral. It iscommon in drug addicts, recipients of blood products, andpatients on haemodialysis. It is also observed commonly inhealth care workers as well as healthy voluntary blooddonors.

Hepatitis C virus (HCV) infection rate is about 3% withmore than 170 million people living with the infection worldover [10]. More than 3.5 million new cases are diagnosedevery year.

Table 1 Year wise distribution of anti-HCV and anti-HBc marker results

Year Total number of Number of donors Number of donors Number of donorsdonations reactive for anti-HCV reactive for anti-HBc positive for both anti-HCV

antibodies by ELISA antibodies by ELISA antibodies by ELISA and anti-HBc2006 18278 75 (0.41%) 2012 (11.01%) 26(0.14%)2007 19664 75 (0.38%) 2013 (10.2%) 16 (0.08%)2008 21068 86 (0.41%) 2098 (9.95%) 15 (0.07%)2009 20605 86 (0.42%) 2145 (10.41%) 22 (0.11%)2010 19515 84 (0.43%) 1901 (9.74%) 17 (0.09%)

TOTAL 99131 406(0. 41%) 10169 (10.25%) 96(0.09%)

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well as HIV infection until the development of advancedscreening tests for anti HCV antibodies and for HCV RNA.Current studies reveal that anti-HBc testing does notidentify additional donors capable of transmitting HCVinfection when such donors are also screened by the moreadvanced and sensitive anti-HCV tests. It has been longargued that anti-HBc testing does help in detection of atleast a few additional donors infected with hepatitis B,which are either in the window period or have a very lowlevel of antigenemia. Despite these potential benefits ofanti-HBc screening, the present test for anti-HBc givesmany false positive results. This not only leads tounnecessary wastage of blood units that are otherwisesuitable for transfusion but also creates unwarrantedanxiety and apprehension among donors who are providedwith confusing test results and are subjected to needlessmedical expense [4].

In our study we detected 96 donors who were positivefor both, anti-HCV and anti-HBc antibodies, 93 of thembeing negative for HBsAg by ELISA. These results can beexplained in several ways. False positive results mayaccount for a handful of such cases. Similarly donationduring the “window period” following acute HBVinfection, remote infection with HBV without persistentviremia, and remote infection with persistent “occult”infection in addition to persistent HCV infection will alsogive such test results.

Nucleic acid testing, however showed presence of HCVRNA only in 42 out of these 96 cases, while HBV DNA wasidentified in 2 cases. The remaining cases where NAT wasnon reactive may indicate either a false positive ELISAresult or a case where HCV viremia has been cleared. There

In this study we detected anti-HCV antibodies byELISA in 406 donors, constituting an average of 0.41% ofthe total. This is fairly comparable to existing Indian[11,12] as well as western literature [13,14] with a fewexceptions [15,16] where a relatively higher prevalencewas reported. Over a period of these five years the HCVprevalence at our centre has remained low with percentagepositivity ranging from 0.38% in 2007 to 0.43% in 2010.These results are possibly due to the strict screening criteriathat are being enforced in our centre, and deferral/rejectionof donors giving positive history of jaundice, high riskbehavior or any other history suggestive of the same.

The transmission of hepatitis B following transfusion ofblood/blood products containing the hepatitis B coreantibody was first described by Hoofnagle in 1978 [17].HBsAg is the most commonly used marker for HBVinfection in donated blood and in many countries includingIndia, anti-HBc is not a mandatory screening test. Anti-HBchas been found to be an excellent indicator of occult HBVinfection during the ‘core window’ period. However, withthe introduction and institution of more advancedtechniques for Nucleic Acid Testing like TMA or PCR,those are able to amplify and detect the HBV DNA, the roleof anti-HBc antibody in donor screening is beingquestioned by users across the globe.

In India, the incidence of anti-HBc amongst blooddonors ranges from 11-29% [18,19]. For reasons essentiallysimilar to those implicated in low HCV prevalence,positivity rates for anti-HBc antibodies in our study areslightly lower, the average being 10.32%.

Anti-HBc was a popular surrogate marker for HCV as

Fig 1 Year wise distribution of anti-HCV and anti-HBC antibody results in blood donors

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301 Apollo Medicine, Vol. 7, No. 4, December 2010

were only four cases (0.004%) where HCV RNA wasidentified in the absence of anti- HCV and presence of antiHBc antibody, 3 of which were also reactive for HBsAg.From these results it is clear that the importance of anti HBcas a surrogate marker for HCV infection cannot beoveremphasized.

As we have already mentioned, patients with HCV-related chronic liver disease (CLD) frequently showmarkers of previous HBV infection. Moreover, they maycarry occult HBV infection. Like dual HBV and HCVinfection, occult HBV infection in chronic hepatitis C couldalso aggravate the disease severity. Suppression of HBVreplication by HCV in acutely or chronically infectedpatients is well-described phenomenon. Liaw, et al [20]found that HCV infection might suppress HBV or eveneliminate HBV. The mechanisms accounting for thesuppression of HCV on HBV were investigated by Shih,et al [21]. Their findings suggest that HCV may directlyinterfere with HBV replication and furthermore identifiedthe HCV core protein as a repressor of HBV production.

HBV-HCV co-infection is characterized by “viralinterference,” whereby the replication of one virus issuppressed by another [22-24]. In the phenomenon of viralinterference, usually one virus remains dormant while theother replicates actively. HCV replication is generally moredominant, leading to low levels of HBV DNA serum[22,26-28] and liver [20,29].

CONCLUSION

The prevalence of anti-HCV and anti-HBc antibodiesindividually are fairly comparable to the existing Indian andwestern literature. Cases positive for both these antibodiesbut lacking HBsAg may indicate dual HBV and HCVinfection, chronic HCV infection with occult Hepatitis B ora false negative result among others. Besides in this era ofhighly sensitive ELISA and NAT, the role of the hepatitis Bcore antibody as a surrogate HCV marker is doubtful.

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