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Prevalence of anti-HCV Antibodies Among Healthy Asymptomatic Indian Blood Donors and the Current Role of anti-HBc Screening as a Surrogate Marker for HCV Infection
Original Article
PREVALENCE OF ANTI-HCV ANTIBODIES AMONG HEALTHY ASYMPTOMATIC INDIAN BLOOD 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 liver diseases world-wide. Since, both the viruses share similar risk factors and modes of transmission, a combined HBV and HCV infection is frequently encountered especially in the HBV endemic areas. Until lately anti-HBc antibodies were considered as surrogate marker for HCV infection. But with the development of advanced tests 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.
INTRODUCTION It is the prime responsibility of a Blood Transfusion Service (BTS) facility to offer its patients the safest possible blood for transfusion that is free from various Transfusion Transmissible Infections (TTI’s). Although measures such as adoption of strict donor selection criteria, encouragement and maintenance voluntary non remunerative pool of blood donors and temporary or permanent deferral of those with high risk behaviors judged by the use of questionnaire are a routine practice globally, the final decision on whether or not to use a blood/blood product for transfusion relies on the results of infectious marker tests. In India, it is mandatory to test for HIV, HBsAg, anti HCV, Malarial Antigen and syphilis. The use of sensitive screening methods that may detect infectious agents during the ‘window period’ such as nucleic 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 liver diseases world-wide [1-2]. Since, both the viruses share similar risk factors and modes of transmission, a combined HBV and HCV infection is frequently encountered especially in the HBV endemic areas. It has been shown that a dual HBV and HCV infection is associated with a more severe liver disease, and is at an increased risk for progression to hepatocellular carcinoma (HCC) [3]. Individual literature on the prevalence of HCV as well as the Hepatitis B core antibody in different populations world over is readily available, however not many have studied the two in conjunction with each other. Presence of 298
anti-HCV and anti-HBc antibodies in absence of detectable HBsAg by ELISA has been reported in blood donors in variable frequencies. Although blood units in such cases are definitely discarded, these cases create a dilemma where donor notification is concerned. Until lately anti-HBc antibodies were considered as surrogate marker for HCV infection. But with the development of advanced tests for HCV detection the role of anti-HBc in this 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 has been shown that co-infection with hepatitis delta virus or HCV results in down regulation of HBV replication and a reduction in antigen synthesis [5]. It was initially believed that the HCV core protein inhibits HBV replication and gene expression.[6] Bellecave, et al. [7] on the contrary showed that HBV and HCV can replicate in the same cell line without in vitro interference. Thereafter other indirect mechanisms 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 Indraprastha Medical Corporation Limited Blood Bank, Indraprastha Apollo Hospital, New Delhi from January 2006 to December 2010 were enrolled in the study. The donors were apparently healthy adults between the ages of 18-60 years. All donors were subjected to a pre test counseling which was done by qualified staff trained to screen donors Apollo Medicine, Vol. 7, No. 4, December 2010
Original Article
for blood donation as per the Drug and Cosmetics act. Donors who did not fulfill the general criteria for blood donation, paid and commercial donors and those with a history of high risk behavior were deferred from donating blood at our institution and hence, excluded from the study. Post donation all donor blood samples were tested for the presence of anti-HBc antibodies using Enzyme Linked Immuno Sorbent Assay (ELISA). Third generation ELISA kits (MonolisaTM Anti-HBc PLUS, BIO-RAD) using fully automated EVOLIS walk away system (BIO-RAD) were used for testing. For anti-HCV antibodies, third generation ELISA kits (MUREX ANTI-HCV (VERSION 4) MUREX BIOTECH.) were used from January 2006 till October 2010. From October 2010 till December 2010 SPNANBASE C-96 3.0 kits from GENERAL BIOLOGICALS CORP. were introduced. All samples that tested positive by ELISA were repeat tested in duplicate using the same ELISA kit. Only repeat reactive samples were labeled as ‘POSITIVE’. Nucleic acid testing (NAT) was done for all donor samples using the TMA (Transcription Mediated Amplification). The Procleix Ultrio Assay, a qualitative in vitro nucleic acid amplification test from Chiron (Novartis), for the detection of human immunodeficiency virus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA, and hepatitis B virus (HBV) DNA was performed and The Procleix HIV-1, HCV, and HBV Discriminatory Assays were run for the reactive cases. ELISA and NAT results were compiled, tabulated and analyzed.
From 2006 till 2010, anti-HCV antibodies were detected in 406 donors, with an average of 81 cases per year. These constitute an average of 0.41% of the total donors tested. Anti-HBc antibody on the other hand was detected in 10,169 donors (average of 10.25%) irrespective of the HBsAg status. Ninety Six (0.09%) showed positivity for both anti-HCV and anti-HBc antibodies. In three out of these 96 donors HBsAg was detected indicating a clear cut case of dual infection of HBV and HCV. However, in the remaining 93 donors only anti-HCV and anti-HBc antibodies were detected (Fig.1). All the 96 cases were subjected to NAT. The initial Procleix Ultrio assay was reactive in 44 cases. Discriminatory assays confirmed the presence of HCV RNA in 42 out of these 44 cases, while the remaining two were diagnosed having HBV DNA. Retrospective analysis revealed that in one of these two cases HBsAg was detected in ELISA, while the other one was only positive for anti HBc antibody besides Anti HCV. A retrospective evaluation of the NAT positive cases also revealed that there were merely 4 cases in the five year study period where anti HBc positivity in absence of anti HCV yielded HCV RNA in NAT. Further in 3 out of these four ELISA also showed HBsAg positivity. DISCUSSION
RESULTS
Hepatitis C is a leading cause of transfusion related hepatits, the transmission primarily being parenteral. It is common in drug addicts, recipients of blood products, and patients on haemodialysis. It is also observed commonly in health care workers as well as healthy voluntary blood donors.
A total of 99,131 donors were included in the study and tested for anti-HCV and anti-HBc antibodies by ELISA. The year wise distribution of blood donors and marker positivity is shown in Table1.
Hepatitis C virus (HCV) infection rate is about 3% with more than 170 million people living with the infection world over [10]. More than 3.5 million new cases are diagnosed every year.
Table 1 Year wise distribution of anti-HCV and anti-HBc marker results Year
Total number of donations
Number of donors reactive for anti-HCV antibodies by ELISA
Number of donors reactive for anti-HBc antibodies by ELISA
Number of donors positive for both anti-HCV antibodies by ELISA and anti-HBc
2006
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%)
406(0. 41%)
10169 (10.25%)
96(0.09%)
TOTAL 99131
299
Apollo Medicine, Vol. 7, No. 4, December 2010
Original Article
Fig 1 Year wise distribution of anti-HCV and anti-HBC antibody results in blood donors
In this study we detected anti-HCV antibodies by ELISA in 406 donors, constituting an average of 0.41% of the total. This is fairly comparable to existing Indian [11,12] as well as western literature [13,14] with a few exceptions [15,16] where a relatively higher prevalence was reported. Over a period of these five years the HCV prevalence at our centre has remained low with percentage positivity ranging from 0.38% in 2007 to 0.43% in 2010. These results are possibly due to the strict screening criteria that are being enforced in our centre, and deferral/rejection of donors giving positive history of jaundice, high risk behavior or any other history suggestive of the same. The transmission of hepatitis B following transfusion of blood/blood products containing the hepatitis B core antibody was first described by Hoofnagle in 1978 [17]. HBsAg is the most commonly used marker for HBV infection in donated blood and in many countries including India, anti-HBc is not a mandatory screening test. Anti-HBc has been found to be an excellent indicator of occult HBV infection during the ‘core window’ period. However, with the introduction and institution of more advanced techniques for Nucleic Acid Testing like TMA or PCR, those are able to amplify and detect the HBV DNA, the role of anti-HBc antibody in donor screening is being questioned by users across the globe. In India, the incidence of anti-HBc amongst blood donors ranges from 11-29% [18,19]. For reasons essentially similar to those implicated in low HCV prevalence, positivity rates for anti-HBc antibodies in our study are slightly lower, the average being 10.32%. Anti-HBc was a popular surrogate marker for HCV as Apollo Medicine, Vol. 7, No. 4, December 2010
well as HIV infection until the development of advanced screening tests for anti HCV antibodies and for HCV RNA. Current studies reveal that anti-HBc testing does not identify additional donors capable of transmitting HCV infection when such donors are also screened by the more advanced and sensitive anti-HCV tests. It has been long argued that anti-HBc testing does help in detection of at least a few additional donors infected with hepatitis B, which are either in the window period or have a very low level of antigenemia. Despite these potential benefits of anti-HBc screening, the present test for anti-HBc gives many false positive results. This not only leads to unnecessary wastage of blood units that are otherwise suitable for transfusion but also creates unwarranted anxiety and apprehension among donors who are provided with confusing test results and are subjected to needless medical expense [4]. In our study we detected 96 donors who were positive for both, anti-HCV and anti-HBc antibodies, 93 of them being negative for HBsAg by ELISA. These results can be explained in several ways. False positive results may account for a handful of such cases. Similarly donation during the “window period” following acute HBV infection, remote infection with HBV without persistent viremia, and remote infection with persistent “occult” infection in addition to persistent HCV infection will also give such test results. Nucleic acid testing, however showed presence of HCV RNA only in 42 out of these 96 cases, while HBV DNA was identified in 2 cases. The remaining cases where NAT was non reactive may indicate either a false positive ELISA result or a case where HCV viremia has been cleared. There
300
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were only four cases (0.004%) where HCV RNA was identified in the absence of anti- HCV and presence of anti HBc antibody, 3 of which were also reactive for HBsAg. From these results it is clear that the importance of anti HBc as a surrogate marker for HCV infection cannot be overemphasized.
12(27): 4406-4410. 4. Infectious Disease Testing for Blood Transfusions. National Institutes of Health Consensus Development Conference Statement. January 9-12, 1995. 5. Weinberger KM, Bauer T, Bohm S, Jilg WG. High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum. J Gen Virol. 2000; 81:1165–1174.
As we have already mentioned, patients with HCVrelated chronic liver disease (CLD) frequently show markers of previous HBV infection. Moreover, they may carry occult HBV infection. Like dual HBV and HCV infection, occult HBV infection in chronic hepatitis C could also aggravate the disease severity. Suppression of HBV replication by HCV in acutely or chronically infected patients is well-described phenomenon. Liaw, et al [20] found that HCV infection might suppress HBV or even eliminate HBV. The mechanisms accounting for the suppression of HCV on HBV were investigated by Shih, et al [21]. Their findings suggest that HCV may directly interfere with HBV replication and furthermore identified the HCV core protein as a repressor of HBV production. HBV-HCV co-infection is characterized by “viral interference,” whereby the replication of one virus is suppressed by another [22-24]. In the phenomenon of viral interference, usually one virus remains dormant while the other replicates actively. HCV replication is generally more dominant, leading to low levels of HBV DNA serum [22,26-28] and liver [20,29]. CONCLUSION The prevalence of anti-HCV and anti-HBc antibodies individually are fairly comparable to the existing Indian and western literature. Cases positive for both these antibodies but lacking HBsAg may indicate dual HBV and HCV infection, chronic HCV infection with occult Hepatitis B or a false negative result among others. Besides in this era of highly sensitive ELISA and NAT, the role of the hepatitis B core antibody as a surrogate HCV marker is doubtful. REFERENCES 1. Lee DS, Huh K, Lee EH, Lee DH, Hong KS, Sung YC. HCV and HBV coexist in HBsAg-negative patients with HCV viremia: possibility of co-infection in these patients must be considered in HBV-high endemic area. J Gastroenterol Hepatol 1997; 12: 855-861. 2. Zarski JP, Bohn B, Bastie A, Pawlotsky JM, Baud M, BostBezeaux F, Tran van Nhieu J, Seigneurin JM, Buffet C, Dhumeaux D. Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 1998; 28: 27-33. 3. Ahmed Helmy, Mohammed Ibrahim Al-Sebayel. Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection. World J Gastroenterol 2006,
6. Pasquinelli C, Shoenberger JM, Chung J, et al. Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology 1997; 25: 719-727. 7. Bellecave P, Gouttenoire J, Gajer M, et al. Hepatitis B and C virus coinfection: a novel model system reveals the absence of direct viral interference. Hepatology. 2009; 50: 46-55. 8. Mimms LT, Mosley JW, Hollinger FB, et al. Effect of concurrent acute infection with hepatitis C virus on acute hepatitis B virus infection. BMJ. 1993; 307: 1095-1097. 9. Chu CJ, Lee SD. Hepatitis B virus/hepatitis C virus coinfection: epidemiology, clinical features, viral interactions and treatment. J Gastroenterol Hepatol 2008; 23: 512-520. 10. Lavanchy D. The global burden of hepatitis C. Liver Int. 2009; 29 (Suppl 1): 74-81. 11. Makroo RN, Raina V, Kaushik V. Prevalence of Hepatitis C Virus antibodies in healthy blood donors. Ind J Med Res. 1999; 110: 123-125 12. Chaudhury N, Ramesh V, Saraswati S, Naik S, et al. Effectiveness of mandatory transmissible disease screening in Indian blood donors. Ind J Med Res. 1995; 101: 229-232. 13. Kuhnl P, Seidl S, Stangel W, Beyer J, Sibrowski W, Flik J, et al. Antibodies to Hepatitis C virus in German blood donors. Lancet. 1989; 324-338. 14. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science, 1989; 244: 362-364. 15. Sumathy S, Valliammai T, Thyagrajan SP, Malathy S, Madangopalan N, Sankaranayaranan V, et al. Prevalence of hepatitis C virus infection in liver diseases, renal diseases and voluntary blood donors in South India. Ind J Med Microbiol. 1993; 11: 291-297. 16. Ghuman HK. Detection of Hepatitis C virus by 3rd generation enzyme immunoassay (lett.). Ind J Gastroentrol 1995; 14: 154. 17. Hoofnagle J H, Seef LB, Bales ZB, et al. Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. N Engl J Med 1978 : 298: 13791383. 18. Kant L, Arora NK. Transmission of hepatitis B virus in
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Original Article children: Indian scenario. In: Sarin SK, Singhal AK, editors. Hepatitis B in India: Problems and prevention. 1st ed. Delhi: CBS publications; 1996. 21-32. 19. Makroo RN. Effectiveness of Screening Blood for Anti HBc & Anti HCV on post Transfusion Hepatitis in Multiply Transfused Patients. Indian Journal of Hematology & Blood Transfusion. 2001, 19 (1); 49-50. 20. Liaw YF, Tsai SL, Chang JJ, Sheen IS, Chien RN, Lin DY, Chu CM. Displacement of hepatitis B virus by hepatitis C virus as the cause of continuing chronic hepatitis. Gastroenterology. 1994;106(4):1048-1053. 21. Shih CM, Lo SJ, Miyamura T, Chen SY, Lee YH. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells. J Virol. 1993; 67(10): 5823-5832. 22. Jardi R, Rodriguez F, Buti M, Costa X, Cotrina M, Galimany R, Esteban R, Guardia J. Role of hepatitis B, C, and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal core promoter mutations on viral replicative interference. Hepatology. 2001; 34: 404-410. 23. Pontisso P., Gerotto M, Benvegnu L, Chemello L, Alberti A. Coinfection by hepatitis B virus and hepatitis C virus. Antivir. Ther. 1998; 3: 137-142.
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24. Sagnelli E, Coppola N, Scolastico C, Filippini P, Santantonio T, Stroffolini T, Piccinino F. Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis. Hepatology. 2000; 32: 1106-1110 25. Brotman B, Prince A M, Huima T, Richardson L, van den Ende MC, Pfeifer U. Interference between non-A, non-B and hepatitis B virus infection in chimpanzees. J Med Virol. 1983; 11: 191-205. 26. Crespo J, Lozano JL, de la Cruz F, Rodrigo L, Rodriguez M, San Miguel G, Artinano E, Pons-Romero F. Prevalence and significance of hepatitis C viremia in chronic active hepatitis B Am J Gastroenterol. 1994; 89: 1147-1151. 27. Koike K, Yasuda K, Yotsuyanagi H, Moriya K, Hino K, Kurokawa K, Iino S. Dominant replication of either virus in dual infection with hepatitis viruses B and C. J Med Virol 1995; 45: 236-239. 28. Liaw YF. Role of hepatitis C virus in dual and triple hepatitis virus infection. Hepatology. 1995; 22: 1101-1108. 29. Yap SH, Hellings JA, Rijntjes PJ, van Loon AM, Duermeyer W, Stute R. Absence of detectable hepatitis B virus DNA in sera and liver of chimpanzees with non-A, non-B hepatitis. J Med Virol. 1985; 15: 343-350.
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PREVALENCE OF ANTI-HCV ANTIBODIES AMONG HEALTHY ASYMPTOMATIC INDIAN BLOOD 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 liver diseases world-wide. Since, both the viruses share similar risk factors and modes of transmission, a combined HBV and HCV infection is frequently encountered especially in the HBV endemic areas. Until lately anti-HBc antibodies were considered as surrogate marker for HCV infection. But with the development of advanced tests 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.
INTRODUCTION It is the prime responsibility of a Blood Transfusion Service (BTS) facility to offer its patients the safest possible blood for transfusion that is free from various Transfusion Transmissible Infections (TTI’s). Although measures such as adoption of strict donor selection criteria, encouragement and maintenance voluntary non remunerative pool of blood donors and temporary or permanent deferral of those with high risk behaviors judged by the use of questionnaire are a routine practice globally, the final decision on whether or not to use a blood/blood product for transfusion relies on the results of infectious marker tests. In India, it is mandatory to test for HIV, HBsAg, anti HCV, Malarial Antigen and syphilis. The use of sensitive screening methods that may detect infectious agents during the ‘window period’ such as nucleic 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 liver diseases world-wide [1-2]. Since, both the viruses share similar risk factors and modes of transmission, a combined HBV and HCV infection is frequently encountered especially in the HBV endemic areas. It has been shown that a dual HBV and HCV infection is associated with a more severe liver disease, and is at an increased risk for progression to hepatocellular carcinoma (HCC) [3]. Individual literature on the prevalence of HCV as well as the Hepatitis B core antibody in different populations world over is readily available, however not many have studied the two in conjunction with each other. Presence of 298
anti-HCV and anti-HBc antibodies in absence of detectable HBsAg by ELISA has been reported in blood donors in variable frequencies. Although blood units in such cases are definitely discarded, these cases create a dilemma where donor notification is concerned. Until lately anti-HBc antibodies were considered as surrogate marker for HCV infection. But with the development of advanced tests for HCV detection the role of anti-HBc in this 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 has been shown that co-infection with hepatitis delta virus or HCV results in down regulation of HBV replication and a reduction in antigen synthesis [5]. It was initially believed that the HCV core protein inhibits HBV replication and gene expression.[6] Bellecave, et al. [7] on the contrary showed that HBV and HCV can replicate in the same cell line without in vitro interference. Thereafter other indirect mechanisms 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 Indraprastha Medical Corporation Limited Blood Bank, Indraprastha Apollo Hospital, New Delhi from January 2006 to December 2010 were enrolled in the study. The donors were apparently healthy adults between the ages of 18-60 years. All donors were subjected to a pre test counseling which was done by qualified staff trained to screen donors Apollo Medicine, Vol. 7, No. 4, December 2010
Original Article
for blood donation as per the Drug and Cosmetics act. Donors who did not fulfill the general criteria for blood donation, paid and commercial donors and those with a history of high risk behavior were deferred from donating blood at our institution and hence, excluded from the study. Post donation all donor blood samples were tested for the presence of anti-HBc antibodies using Enzyme Linked Immuno Sorbent Assay (ELISA). Third generation ELISA kits (MonolisaTM Anti-HBc PLUS, BIO-RAD) using fully automated EVOLIS walk away system (BIO-RAD) were used for testing. For anti-HCV antibodies, third generation ELISA kits (MUREX ANTI-HCV (VERSION 4) MUREX BIOTECH.) were used from January 2006 till October 2010. From October 2010 till December 2010 SPNANBASE C-96 3.0 kits from GENERAL BIOLOGICALS CORP. were introduced. All samples that tested positive by ELISA were repeat tested in duplicate using the same ELISA kit. Only repeat reactive samples were labeled as ‘POSITIVE’. Nucleic acid testing (NAT) was done for all donor samples using the TMA (Transcription Mediated Amplification). The Procleix Ultrio Assay, a qualitative in vitro nucleic acid amplification test from Chiron (Novartis), for the detection of human immunodeficiency virus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA, and hepatitis B virus (HBV) DNA was performed and The Procleix HIV-1, HCV, and HBV Discriminatory Assays were run for the reactive cases. ELISA and NAT results were compiled, tabulated and analyzed.
From 2006 till 2010, anti-HCV antibodies were detected in 406 donors, with an average of 81 cases per year. These constitute an average of 0.41% of the total donors tested. Anti-HBc antibody on the other hand was detected in 10,169 donors (average of 10.25%) irrespective of the HBsAg status. Ninety Six (0.09%) showed positivity for both anti-HCV and anti-HBc antibodies. In three out of these 96 donors HBsAg was detected indicating a clear cut case of dual infection of HBV and HCV. However, in the remaining 93 donors only anti-HCV and anti-HBc antibodies were detected (Fig.1). All the 96 cases were subjected to NAT. The initial Procleix Ultrio assay was reactive in 44 cases. Discriminatory assays confirmed the presence of HCV RNA in 42 out of these 44 cases, while the remaining two were diagnosed having HBV DNA. Retrospective analysis revealed that in one of these two cases HBsAg was detected in ELISA, while the other one was only positive for anti HBc antibody besides Anti HCV. A retrospective evaluation of the NAT positive cases also revealed that there were merely 4 cases in the five year study period where anti HBc positivity in absence of anti HCV yielded HCV RNA in NAT. Further in 3 out of these four ELISA also showed HBsAg positivity. DISCUSSION
RESULTS
Hepatitis C is a leading cause of transfusion related hepatits, the transmission primarily being parenteral. It is common in drug addicts, recipients of blood products, and patients on haemodialysis. It is also observed commonly in health care workers as well as healthy voluntary blood donors.
A total of 99,131 donors were included in the study and tested for anti-HCV and anti-HBc antibodies by ELISA. The year wise distribution of blood donors and marker positivity is shown in Table1.
Hepatitis C virus (HCV) infection rate is about 3% with more than 170 million people living with the infection world over [10]. More than 3.5 million new cases are diagnosed every year.
Table 1 Year wise distribution of anti-HCV and anti-HBc marker results Year
Total number of donations
Number of donors reactive for anti-HCV antibodies by ELISA
Number of donors reactive for anti-HBc antibodies by ELISA
Number of donors positive for both anti-HCV antibodies by ELISA and anti-HBc
2006
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%)
406(0. 41%)
10169 (10.25%)
96(0.09%)
TOTAL 99131
299
Apollo Medicine, Vol. 7, No. 4, December 2010
Original Article
Fig 1 Year wise distribution of anti-HCV and anti-HBC antibody results in blood donors
In this study we detected anti-HCV antibodies by ELISA in 406 donors, constituting an average of 0.41% of the total. This is fairly comparable to existing Indian [11,12] as well as western literature [13,14] with a few exceptions [15,16] where a relatively higher prevalence was reported. Over a period of these five years the HCV prevalence at our centre has remained low with percentage positivity ranging from 0.38% in 2007 to 0.43% in 2010. These results are possibly due to the strict screening criteria that are being enforced in our centre, and deferral/rejection of donors giving positive history of jaundice, high risk behavior or any other history suggestive of the same. The transmission of hepatitis B following transfusion of blood/blood products containing the hepatitis B core antibody was first described by Hoofnagle in 1978 [17]. HBsAg is the most commonly used marker for HBV infection in donated blood and in many countries including India, anti-HBc is not a mandatory screening test. Anti-HBc has been found to be an excellent indicator of occult HBV infection during the ‘core window’ period. However, with the introduction and institution of more advanced techniques for Nucleic Acid Testing like TMA or PCR, those are able to amplify and detect the HBV DNA, the role of anti-HBc antibody in donor screening is being questioned by users across the globe. In India, the incidence of anti-HBc amongst blood donors ranges from 11-29% [18,19]. For reasons essentially similar to those implicated in low HCV prevalence, positivity rates for anti-HBc antibodies in our study are slightly lower, the average being 10.32%. Anti-HBc was a popular surrogate marker for HCV as Apollo Medicine, Vol. 7, No. 4, December 2010
well as HIV infection until the development of advanced screening tests for anti HCV antibodies and for HCV RNA. Current studies reveal that anti-HBc testing does not identify additional donors capable of transmitting HCV infection when such donors are also screened by the more advanced and sensitive anti-HCV tests. It has been long argued that anti-HBc testing does help in detection of at least a few additional donors infected with hepatitis B, which are either in the window period or have a very low level of antigenemia. Despite these potential benefits of anti-HBc screening, the present test for anti-HBc gives many false positive results. This not only leads to unnecessary wastage of blood units that are otherwise suitable for transfusion but also creates unwarranted anxiety and apprehension among donors who are provided with confusing test results and are subjected to needless medical expense [4]. In our study we detected 96 donors who were positive for both, anti-HCV and anti-HBc antibodies, 93 of them being negative for HBsAg by ELISA. These results can be explained in several ways. False positive results may account for a handful of such cases. Similarly donation during the “window period” following acute HBV infection, remote infection with HBV without persistent viremia, and remote infection with persistent “occult” infection in addition to persistent HCV infection will also give such test results. Nucleic acid testing, however showed presence of HCV RNA only in 42 out of these 96 cases, while HBV DNA was identified in 2 cases. The remaining cases where NAT was non reactive may indicate either a false positive ELISA result or a case where HCV viremia has been cleared. There
300
Original Article
were only four cases (0.004%) where HCV RNA was identified in the absence of anti- HCV and presence of anti HBc antibody, 3 of which were also reactive for HBsAg. From these results it is clear that the importance of anti HBc as a surrogate marker for HCV infection cannot be overemphasized.
12(27): 4406-4410. 4. Infectious Disease Testing for Blood Transfusions. National Institutes of Health Consensus Development Conference Statement. January 9-12, 1995. 5. Weinberger KM, Bauer T, Bohm S, Jilg WG. High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum. J Gen Virol. 2000; 81:1165–1174.
As we have already mentioned, patients with HCVrelated chronic liver disease (CLD) frequently show markers of previous HBV infection. Moreover, they may carry occult HBV infection. Like dual HBV and HCV infection, occult HBV infection in chronic hepatitis C could also aggravate the disease severity. Suppression of HBV replication by HCV in acutely or chronically infected patients is well-described phenomenon. Liaw, et al [20] found that HCV infection might suppress HBV or even eliminate HBV. The mechanisms accounting for the suppression of HCV on HBV were investigated by Shih, et al [21]. Their findings suggest that HCV may directly interfere with HBV replication and furthermore identified the HCV core protein as a repressor of HBV production. HBV-HCV co-infection is characterized by “viral interference,” whereby the replication of one virus is suppressed by another [22-24]. In the phenomenon of viral interference, usually one virus remains dormant while the other replicates actively. HCV replication is generally more dominant, leading to low levels of HBV DNA serum [22,26-28] and liver [20,29]. CONCLUSION The prevalence of anti-HCV and anti-HBc antibodies individually are fairly comparable to the existing Indian and western literature. Cases positive for both these antibodies but lacking HBsAg may indicate dual HBV and HCV infection, chronic HCV infection with occult Hepatitis B or a false negative result among others. Besides in this era of highly sensitive ELISA and NAT, the role of the hepatitis B core antibody as a surrogate HCV marker is doubtful. REFERENCES 1. Lee DS, Huh K, Lee EH, Lee DH, Hong KS, Sung YC. HCV and HBV coexist in HBsAg-negative patients with HCV viremia: possibility of co-infection in these patients must be considered in HBV-high endemic area. J Gastroenterol Hepatol 1997; 12: 855-861. 2. Zarski JP, Bohn B, Bastie A, Pawlotsky JM, Baud M, BostBezeaux F, Tran van Nhieu J, Seigneurin JM, Buffet C, Dhumeaux D. Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 1998; 28: 27-33. 3. Ahmed Helmy, Mohammed Ibrahim Al-Sebayel. Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection. World J Gastroenterol 2006,
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