Significance of HBV NAT Among HBs Antigen–Negative Blood Donors in Saudi Arabia

Significance of HBV NAT Among HBs Antigen–Negative Blood Donors in Saudi Arabia Abstract Background Bloodborne hepatitis B virus (HBV) transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg), or from donors with chronic infections in whom serological markers were not detected, could cause residual infections leading to relevant transfusion-transmitted infections (RTTIs). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total hepatitis B core antibody (anti-HBc)–negative window period of infection and in chronic cases. Objective To assess the presence or absence of HBV DNA in blood donors with HBsAg negativity. Methods We collected 3014 blood specimens from volunteer blood donors at the blood bank of King Khalid University Hospital in Riyadh, Saudia Arabia. Specimens from each donor were tested for HBsAg, anti-HBc, and hepatitis B surface antibody (anti-HBs) by commercial immunoassays and for qualitative assessments of HBV-DNA by HBV-NAT testing. Results Of the 3014 donors, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 for HBsAg (0.03%) only, and of those 264 donors (8.8%) for anti-HBc. Of these last, 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% of subjects who tested HBsAg positive, and nonreactive in 100% of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). Among 2742 donors who tested seronegative, 1 specimen was determined to be reactive via HBV-NAT testing. Conclusions The frequency of HBV DNA in blood donors who tested seronegative was low. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection. hepatitis B virus (HBV), hepatitis B virus nucleic acid testing (HBV NAT), blood donors, HBs Ag, anti-HBc, anti-HBs Hepatitis B virus (HBV) is one of several viruses known to cause viral hepatitis. HBV infection leads to a wide range of liver diseases, such as acute hepatitis (including fulminant hepatic failure), chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC).1 More than two billion people throughout the world have been infected with HBV, of whom more than 350 million are chronically infected carriers.1 Bloodborne HBV transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg; ie, donors in the window period [WP]), or from donors with chronic infections in whom serological markers were not detected, could be the cause of residual infections leading to relevant transfusion-transmitted infections (RTTI). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total HBV core antibody (anti-HBc)–negative WP of infection and in chronic cases. As a result, NAT for HBV has become a routine part of blood-donor infectious-disease screening. Routine HBV NAT screening of blood donations was introduced in Germany in 1997. Since 1997, HBV NAT has been introduced as a routine screening assay in many other countries worldwide, resulting in reduced HBV prevalence levels.2-11 Routine HBV NAT screening of blood donations in Saudi Arabia was introduced in 2008, to complete the screening program for transfusion-relevant viruses. Since the introduction of HBV-NAT, a number of potential WP donations have been identified.8-12 The objective of this study was to assess the presence or absence of HBV DNA in blood specimens from donors who have tested HBsAg negative. Materials and Methods Study Site and Duration A total of 3014 blood specimens were collected from male blood donors at the blood bank of King Khalid Hospital, Al-Majma’ah, Saudi Arabia, from November 2015 through March 2017. The reason for that most of blood donors in Saudi Arabia are male as per Saudi cultural practices and rituals. The specimens from donors were screened in the virology laboratory of King Khalid Hospital, Almajma’ah, Saudi Arabia. Written informed consent forms were signed by all participants in the study. All research procedures have been approved by the National Ethical Committee, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia (MUREC-Feb. 19/COM-201710). All procedures performed in this study were in accordance with the ethical standards of the National Committee of Bioethics and with the 1964 Declaration of Helsinki. After extraction of the fresh serum specimens from the participants, we conducted the following serological tests: HBs Ag testing (ARCHITECT anti-HBc II; Abbott Laboratories Inc.): a one-step immunoassay for the qualitative detection of the HBsAg in human serum and plasma using chemiluminescent microparticle immunoassay (CMIA) technology, with flexible assay protocols that are referred to as Chemiflex Anti-HBc assay (ARCHITECT anti-HBs assay; Abbott Laboratories Inc.): a 2-step immunoassay for the qualitative detection of anti-HBc in human serum and plasma using CMIA technology Hepatitis B surface antigen antibody assay (anti-HBs; ARCHITECT; Abbott Laboratories Inc.): a 2-step immunoassay, using CMIA technology, for the quantitative determination of anti-HBs in human serum and plasma Individual NAT NAT was performed, after extraction of fresh plasma specimens from study participants, by using COBAS Taq Screen MPX (all COBAS products by F. Hoffman-La Roche, Ltd.), a qualitative multiplex test that enables the screening and simultaneous detection of HBV DNA in individual plasma specimens from blood donors. This test uses a generic nucleic-acid preparation technique on the COBAS Ampliprep instrument. HBV DNA is amplified and detected using automated, real-time polymerase chain reaction (PCR) on the COBAS Taq Man Analyzer. All blood donors were tested for NAT. Anti-HBs test was performed for all blood donors who tested anti-HBc positive/HBsAg negative. Individual plasma specimens from blood donors were used for individual NAT testing. Individual NAT testing was done just. We confirmed the HBsAg-positive status (using NAT) of donors via PCR which was performed according to manufacturer specifications. We did not perform real-time quantitative PCR (rt-qPCR). Statistical Analysis Data were analyzed by using SPSS (Statistical Package for the Social Sciences) software, version 17.0. Results The cumulative results of HBsAg and anti-HBc testing on the 3014 blood donors in our cohort are depicted in Figure 1. A total of 272 donors (9.0%) were tested for seroprevalence of HBV infection, of which 8 (0.26%) tested positive for HBsAg and 264 (8.8%) tested positive for anti-HBc and negative for HBsAg. Among the 264 donors who tested anti-HBc positive and HBsAg negative, the most frequent pattern was the association of anti-HBc with anti-HBs, at 78.4% (207/264; Figure 2). Figure 1 View largeDownload slide Cumulative results of surface antigen of the hepatitis B virus (HBsAg) and total hepatitis B core antibody (anti-HBc) testing on blood donors. + indicates positive; −, negative. Figure 1 View largeDownload slide Cumulative results of surface antigen of the hepatitis B virus (HBsAg) and total hepatitis B core antibody (anti-HBc) testing on blood donors. + indicates positive; −, negative. Figure 2 View largeDownload slide Serological patterns of hepatitis B virus (HBV) markers in 264 blood donors who tested negative for surface antigen of the hepatitis B virus (HBsAg) and positive for hepatitis B core antibody (anti-HBc) with and without anti-HBs. Figure 2 View largeDownload slide Serological patterns of hepatitis B virus (HBV) markers in 264 blood donors who tested negative for surface antigen of the hepatitis B virus (HBsAg) and positive for hepatitis B core antibody (anti-HBc) with and without anti-HBs. All donated blood specimens were assayed for HBV-DNA using the HBV-NAT test, which is a commercial qualitative target-amplification method. Also, those specimens were validated for blood-donation screening (COBAS Taq Screen MPX test). HBV-NAT testing showed reactivity in 75.0% (6/8) of subjects who tested HBsAg positive. Nonreactivity was revealed in 100% (264/264) of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). As shown in Table 1, the HBV-NAT test was reactive in 75.0% (6/8) of subjects who tested HBsAg positive and those who tested anti-HBc positive. The test was nonreactive in 12.5% (1/8) of subjects who tested HBsAg positive and anti-HBc positive and in 12.5% (1/8) of those who tested HBsAg positive and anti-HBc negative. Table 1. Results of HBV-NAT Testing on 8 Blood Donors Who Tested Positive for HBsAg, According to Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Table 1. Results of HBV-NAT Testing on 8 Blood Donors Who Tested Positive for HBsAg, According to Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large HBV-NAT testing was nonreactive in 100% (207/207) of subjects who tested anti-HBc positive/HBsAg negative who had anti-HBs. The testing was nonreactive in 100% (57/57) of those who tested anti-HBc positive/HBsAg negative and did not have anti-HBs (Table 2). Among 2742 donors with seronegativity (HBs Ag, anti-HBc, and anti-HBs), 1 specimen was reactive via HBV-NAT testing. Table 2. Results of HBV-NAT Testing on 264 Blood Donors Who Tested Positive for Anti-HBc and Negative for HBsAg, According to the Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Table 2. Results of HBV-NAT Testing on 264 Blood Donors Who Tested Positive for Anti-HBc and Negative for HBsAg, According to the Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Discussion The World Health Organization (WHO) has estimated that worldwide, there are still 360 million people who are chronically infected with HBV.13 It has been estimated14 that infections with HBV were responsible for approximately 59% of HCC cases in developing countries. Screening of donated blood for transfusion-transmissible infections represents one of the most important strategies for blood-transfusion safety and availability. The presence of this type of infection among blood donors is rare. In a central region of Saudi Arabia, El-Hazmi15 reported that between the years 2000 and 2002, a total of 1.5% of blood donors had HBV infection. In the Al-Ahsa area, as detailed by Panhotra et al,16 the study results showed that 1.9% of blood donors tested HBsAg positive, 3.2% tested HBcAb positive, and 10.1% had developed HBsAb and HBcAb. The Al-Turaifi study17 demonstrated a decrease in the percentage of blood donors that have HBsAg, HBcAb, or HBsAb and HBcAb, to 0.67%, 0.9%, and 7.04%, respectively. Our study results revealed a decrease in the percentage of blood donors that have HBsAg only, HBsAg and HBcAb, or HBsAb and HBcAb, to 0.03%, 0.23%, and 6.9%, respectively. Of the 3014 donors we studied, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 donor (0.03%) for HBsAg only, and 264 (8.8%) for anti-HBc, of whom 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% (6/8) of subjects who tested HBsAg positive and nonreactive in 100% (264/264) of subjects who tested positive for anti-HBc/negative for HBsAg (with or without anti-HBs). Among 2742 donors who tested seronegative (HBs Ag, anti-HBc, and anti-HBs), 1 specimen was reactive according to HBV-NAT results. The frequency of HBV DNA in blood donors who tested seronegative (HBs Ag, anti-HBc, and anti-HBs) was low. Several studies18-27 have reported the effectiveness of HBV NAT in reducing the residual risk of transfusion-transmitted HBV infection in HBsAg-negative blood donations in regions with low/high prevalence of HBV. Although many studies have been published on the value of HBV NAT intercepting seronegative units, this study may interest in areas of the world where hepatitis B remains endemic. In the current study, we detected HBV DNA in 0.04% of the blood donors who tested HBsAg negative. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection among blood donors who test negative for anti-HBs. This finding may be explained by the fact that Saudi Arabia had achieved significant cultural and economic development during the past 2 decades. Also, the low frequency of HBV DNA in blood donors who test HBsAg negative blood donors may result from the presence of a national hepatitis B prevention program. We hope that our results may be useful to our colleagues in other countries as they seek new methods to strengthen the safety of their blood supply. LM Acknowledgments We thank the Deanship of Scientific Research, Majma’ah University, Riyadh, Saudi Arabia, for the financial support it provided us to conduct this study. Abbreviations HBV hepatitis B virus HCC hepatocellular carcinoma HBsAg surface antigen of HBV WP window period RTTI relevant transfusion-transmitted infections NAT nucleic acid testing anti-HBc total HBV core antibody CMIA chemiluminescent microparticle immunoassay PCR polymerase chain reaction rt-qPCR real-time quantitative PCR SPSS Statistical Package for Social Sciences WHO World Health Organization NA nonapplicable References 1. Hollinger FB, Liang TJ. Hepatitis B virus. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, eds. Fields Virology. 4 . Philadelphia, PA: Lippincott-Raven Publishers; 2001: 2971– 3036. 2. Brojer E, Grabarczyk P, Liszewski G, Mikulska M, Allain JP, Letowska M; Polish Blood Transfusion Service Viral Study Group. Characterization of HBV DNA+/HBsAg− blood donors in Poland identified by triplex NAT. Hepatology . 2006; 44( 6): 1666– 1674. Google Scholar CrossRef Search ADS PubMed  3. Velati C, Romanò L, Fomiatti L, Baruffi L, Zanetti AR; SIMTI Research Group. Impact of nucleic acid testing for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus on the safety of blood supply in Italy: a 6-year survey. Transfusion . 2008; 48( 10): 2205– 2213. Google Scholar CrossRef Search ADS PubMed  4. Margaritis AR, Brown SM, Seed CR, Kiely P, D’Agostino B, Keller AJ. Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA. Transfusion . 2007; 47( 10): 1783– 1793. Google Scholar CrossRef Search ADS PubMed  5. Nantachit N, Thaikruea L, Thongsawat Set al.   Evaluation of a multiplex human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus nucleic acid testing assay to detect viremic blood donors in northern Thailand. Transfusion . 2007; 47( 10): 1803– 1808. Google Scholar CrossRef Search ADS PubMed  6. Li L, Chen PJ, Chen MH, Chak KF, Lin KS, Tsai SJ. A pilot study for screening blood donors in Taiwan by nucleic acid amplification technology: detecting occult hepatitis B virus infections and closing the serologic window period for hepatitis C virus. Transfusion . 2008; 48( 6): 1198– 1206. Google Scholar CrossRef Search ADS PubMed  7. Vermeulen M, Lelie N, Sykes Wet al.   Impact of individual-donation nucleic acid testing on risk of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus transmission by blood transfusion in South Africa. Transfusion . 2009; 49( 6): 1115– 1125. Google Scholar CrossRef Search ADS PubMed  8. Reesink HW, Engelfriet CP, Henn Get al.   Occult hepatitis B infection in blood donors. Vox Sang . 2008; 94( 2): 153– 166. Google Scholar CrossRef Search ADS PubMed  9. Stolz M, Tinguely C, Graziani Met al.   Efficacy of individual nucleic acid amplification testing in reducing the risk of transfusion-transmitted hepatitis B virus infection in Switzerland, a low-endemic region. Transfusion . 2010; 50( 12): 2695– 2706. Google Scholar CrossRef Search ADS PubMed  10. Roth WK, Weber M, Petersen Det al.   NAT for HBV and anti-HBc testing increase blood safety. Transfusion . 2002; 42( 7): 869– 875. Google Scholar CrossRef Search ADS PubMed  11. Sato S, Ohhashi W, Ihara H, Sakaya S, Kato T, Ikeda H. Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion . 2001; 41( 9): 1107– 1113. Google Scholar CrossRef Search ADS PubMed  12. Jilg W, Sieger E, Zachoval R, Schätzl H. Individuals with antibodies against hepatitis B core antigen as the only serological marker for hepatitis B infection: high percentage of carriers of hepatitis B and C virus. J Hepatol . 1995; 23( 1): 14– 20. Google Scholar CrossRef Search ADS PubMed  13. World Health Organization (WHO). Western Pacific regional plan for hepatitis B control through immunization . WHO website. http://www.wpro.who.int/immunization/documents/docs/POA_HepB.pdf. Accessed April 19, 2018. 14. Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer . 2006; 118( 12): 3030– 3044. Google Scholar CrossRef Search ADS PubMed  15. El-Hazmi MM. Prevalence of HBV, HCV, HIV-1, 2 and HTLV-I/II infections among blood donors in a teaching hospital in the Central region of Saudi Arabia. Saudi Med J . 2004; 25( 1): 26– 33. Google Scholar PubMed  16. Panhotra BR, Al-Bahrani A, Ul-Hassan Z. Epidemiology of antibody to hepatitis B core antigen screening among blood donors in Eastern Saudi Arabia. Need to replace the test by HBV DNA testing. Saudi Med J . 2005; 26( 2): 270– 273. Google Scholar PubMed  17. Al-Turaifi H. Evaluation of nucleic acid testing for blood donors: one year study. Int J Blood Transfus Immunohematol . 2015; 5: 19– 25. Google Scholar CrossRef Search ADS   18. Karimi G, Zadsar M, Vafaei N, Sharifi Z, FalahTafti M. Prevalence of antibody to hepatitis B core antigen and hepatitis B virus DNA in HBsAg negative healthy blood donors. Virol J . 2016; 13: 36. Google Scholar CrossRef Search ADS PubMed  19. Findik D, Arslan U, Baykan M. Determination of hepatitis B virus DNA incidence, viral load, and mutations in blood donors with HBsAg and anti-HBs-negative serology and antibodies to hepatitis B core antigen. Eur J Intern Med . 2007; 18( 8): 571– 575. Google Scholar CrossRef Search ADS PubMed  20. Arabaci F, Oldacay M. Investigation of mutant hepatitis B virus in core antibody seropositive cases of blood donor population. J Med Sci . 2008; 8: 316– 320. Google Scholar CrossRef Search ADS   21. Zervou EK, Dalekos GN, Boumba DS, Tsianos EV. Value of anti-HBc screening of blood donors for prevention of HBV infection: results of a 3-year prospective study in Northwestern Greece. Transfusion . 2001; 41( 5): 652– 658. Google Scholar CrossRef Search ADS PubMed  22. Manzini P, Girotto M, Borsotti Ret al.   Italian blood donors with anti-HBc and occult hepatitis B virus infection. Haematologica . 2007; 92( 12): 1664– 1670. Google Scholar CrossRef Search ADS PubMed  23. Asim M, Ali R, Khan LA, Husain SA, Singla R, Kar P. Significance of anti-HBc screening of blood donors and its association with occult hepatitis B virus infection: implications for blood transfusion. Indian J Med Res . 2010; 132: 312– 317. Google Scholar PubMed  24. Said ZN, Sayed MH, Salama IIet al.   Occult hepatitis B virus infection among Egyptian blood donors. World J Hepatol . 2013; 5( 2): 64– 73. Google Scholar CrossRef Search ADS PubMed  25. Yotsuyanagi H, Yasuda K, Moriya Ket al.   Frequent presence of HBV in the sera of HBsAg-negative, anti-HBc-positive blood donors. Transfusion . 2001; 41( 9): 1093– 1099. Google Scholar CrossRef Search ADS PubMed  26. Mahmoud OA, Ghazal AA, Del Metwally S, Elnour AM, Yousif GE. Detection of occult hepatitis B virus infection among blood donors in Sudan. J Egypt Public Health Assoc . 2013; 88: 14– 18. Google Scholar CrossRef Search ADS PubMed  27. Stramer SL, Notari EP, Krysztof DE, Dodd RY. Hepatitis B virus testing by minipool nucleic acid testing: does it improve blood safety? Transfusion . 2013; 53( 10 Pt 2): 2449– 2458. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Significance of HBV NAT Among HBs Antigen–Negative Blood Donors in Saudi Arabia

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Abstract

Abstract Background Bloodborne hepatitis B virus (HBV) transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg), or from donors with chronic infections in whom serological markers were not detected, could cause residual infections leading to relevant transfusion-transmitted infections (RTTIs). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total hepatitis B core antibody (anti-HBc)–negative window period of infection and in chronic cases. Objective To assess the presence or absence of HBV DNA in blood donors with HBsAg negativity. Methods We collected 3014 blood specimens from volunteer blood donors at the blood bank of King Khalid University Hospital in Riyadh, Saudia Arabia. Specimens from each donor were tested for HBsAg, anti-HBc, and hepatitis B surface antibody (anti-HBs) by commercial immunoassays and for qualitative assessments of HBV-DNA by HBV-NAT testing. Results Of the 3014 donors, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 for HBsAg (0.03%) only, and of those 264 donors (8.8%) for anti-HBc. Of these last, 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% of subjects who tested HBsAg positive, and nonreactive in 100% of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). Among 2742 donors who tested seronegative, 1 specimen was determined to be reactive via HBV-NAT testing. Conclusions The frequency of HBV DNA in blood donors who tested seronegative was low. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection. hepatitis B virus (HBV), hepatitis B virus nucleic acid testing (HBV NAT), blood donors, HBs Ag, anti-HBc, anti-HBs Hepatitis B virus (HBV) is one of several viruses known to cause viral hepatitis. HBV infection leads to a wide range of liver diseases, such as acute hepatitis (including fulminant hepatic failure), chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC).1 More than two billion people throughout the world have been infected with HBV, of whom more than 350 million are chronically infected carriers.1 Bloodborne HBV transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg; ie, donors in the window period [WP]), or from donors with chronic infections in whom serological markers were not detected, could be the cause of residual infections leading to relevant transfusion-transmitted infections (RTTI). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total HBV core antibody (anti-HBc)–negative WP of infection and in chronic cases. As a result, NAT for HBV has become a routine part of blood-donor infectious-disease screening. Routine HBV NAT screening of blood donations was introduced in Germany in 1997. Since 1997, HBV NAT has been introduced as a routine screening assay in many other countries worldwide, resulting in reduced HBV prevalence levels.2-11 Routine HBV NAT screening of blood donations in Saudi Arabia was introduced in 2008, to complete the screening program for transfusion-relevant viruses. Since the introduction of HBV-NAT, a number of potential WP donations have been identified.8-12 The objective of this study was to assess the presence or absence of HBV DNA in blood specimens from donors who have tested HBsAg negative. Materials and Methods Study Site and Duration A total of 3014 blood specimens were collected from male blood donors at the blood bank of King Khalid Hospital, Al-Majma’ah, Saudi Arabia, from November 2015 through March 2017. The reason for that most of blood donors in Saudi Arabia are male as per Saudi cultural practices and rituals. The specimens from donors were screened in the virology laboratory of King Khalid Hospital, Almajma’ah, Saudi Arabia. Written informed consent forms were signed by all participants in the study. All research procedures have been approved by the National Ethical Committee, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia (MUREC-Feb. 19/COM-201710). All procedures performed in this study were in accordance with the ethical standards of the National Committee of Bioethics and with the 1964 Declaration of Helsinki. After extraction of the fresh serum specimens from the participants, we conducted the following serological tests: HBs Ag testing (ARCHITECT anti-HBc II; Abbott Laboratories Inc.): a one-step immunoassay for the qualitative detection of the HBsAg in human serum and plasma using chemiluminescent microparticle immunoassay (CMIA) technology, with flexible assay protocols that are referred to as Chemiflex Anti-HBc assay (ARCHITECT anti-HBs assay; Abbott Laboratories Inc.): a 2-step immunoassay for the qualitative detection of anti-HBc in human serum and plasma using CMIA technology Hepatitis B surface antigen antibody assay (anti-HBs; ARCHITECT; Abbott Laboratories Inc.): a 2-step immunoassay, using CMIA technology, for the quantitative determination of anti-HBs in human serum and plasma Individual NAT NAT was performed, after extraction of fresh plasma specimens from study participants, by using COBAS Taq Screen MPX (all COBAS products by F. Hoffman-La Roche, Ltd.), a qualitative multiplex test that enables the screening and simultaneous detection of HBV DNA in individual plasma specimens from blood donors. This test uses a generic nucleic-acid preparation technique on the COBAS Ampliprep instrument. HBV DNA is amplified and detected using automated, real-time polymerase chain reaction (PCR) on the COBAS Taq Man Analyzer. All blood donors were tested for NAT. Anti-HBs test was performed for all blood donors who tested anti-HBc positive/HBsAg negative. Individual plasma specimens from blood donors were used for individual NAT testing. Individual NAT testing was done just. We confirmed the HBsAg-positive status (using NAT) of donors via PCR which was performed according to manufacturer specifications. We did not perform real-time quantitative PCR (rt-qPCR). Statistical Analysis Data were analyzed by using SPSS (Statistical Package for the Social Sciences) software, version 17.0. Results The cumulative results of HBsAg and anti-HBc testing on the 3014 blood donors in our cohort are depicted in Figure 1. A total of 272 donors (9.0%) were tested for seroprevalence of HBV infection, of which 8 (0.26%) tested positive for HBsAg and 264 (8.8%) tested positive for anti-HBc and negative for HBsAg. Among the 264 donors who tested anti-HBc positive and HBsAg negative, the most frequent pattern was the association of anti-HBc with anti-HBs, at 78.4% (207/264; Figure 2). Figure 1 View largeDownload slide Cumulative results of surface antigen of the hepatitis B virus (HBsAg) and total hepatitis B core antibody (anti-HBc) testing on blood donors. + indicates positive; −, negative. Figure 1 View largeDownload slide Cumulative results of surface antigen of the hepatitis B virus (HBsAg) and total hepatitis B core antibody (anti-HBc) testing on blood donors. + indicates positive; −, negative. Figure 2 View largeDownload slide Serological patterns of hepatitis B virus (HBV) markers in 264 blood donors who tested negative for surface antigen of the hepatitis B virus (HBsAg) and positive for hepatitis B core antibody (anti-HBc) with and without anti-HBs. Figure 2 View largeDownload slide Serological patterns of hepatitis B virus (HBV) markers in 264 blood donors who tested negative for surface antigen of the hepatitis B virus (HBsAg) and positive for hepatitis B core antibody (anti-HBc) with and without anti-HBs. All donated blood specimens were assayed for HBV-DNA using the HBV-NAT test, which is a commercial qualitative target-amplification method. Also, those specimens were validated for blood-donation screening (COBAS Taq Screen MPX test). HBV-NAT testing showed reactivity in 75.0% (6/8) of subjects who tested HBsAg positive. Nonreactivity was revealed in 100% (264/264) of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). As shown in Table 1, the HBV-NAT test was reactive in 75.0% (6/8) of subjects who tested HBsAg positive and those who tested anti-HBc positive. The test was nonreactive in 12.5% (1/8) of subjects who tested HBsAg positive and anti-HBc positive and in 12.5% (1/8) of those who tested HBsAg positive and anti-HBc negative. Table 1. Results of HBV-NAT Testing on 8 Blood Donors Who Tested Positive for HBsAg, According to Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Table 1. Results of HBV-NAT Testing on 8 Blood Donors Who Tested Positive for HBsAg, According to Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag positive Anti-HBc positive Anti-HBs negative  6 (75.0%)  1 (12.5%)  HBs Ag positive Anti-HBc negative Anti-HBs negative  NA  1 (12.5%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large HBV-NAT testing was nonreactive in 100% (207/207) of subjects who tested anti-HBc positive/HBsAg negative who had anti-HBs. The testing was nonreactive in 100% (57/57) of those who tested anti-HBc positive/HBsAg negative and did not have anti-HBs (Table 2). Among 2742 donors with seronegativity (HBs Ag, anti-HBc, and anti-HBs), 1 specimen was reactive via HBV-NAT testing. Table 2. Results of HBV-NAT Testing on 264 Blood Donors Who Tested Positive for Anti-HBc and Negative for HBsAg, According to the Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Table 2. Results of HBV-NAT Testing on 264 Blood Donors Who Tested Positive for Anti-HBc and Negative for HBsAg, According to the Serological Profile Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  Serological Profile  HBV NAT  No. (%)  Reactive  Nonreactive  HBs Ag negative Anti-HBc positive Anti-HBs positive  NA  207 (100%)  HBs Ag negative Anti-HBc positive Anti-HBs negative  NA  57 (100%)  HBV, hepatitis B virus; NAT, nucleic acid testing; HBs Ag, surface antigen of HBV; anti-HBc, total hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; NA, nonapplicable. View Large Discussion The World Health Organization (WHO) has estimated that worldwide, there are still 360 million people who are chronically infected with HBV.13 It has been estimated14 that infections with HBV were responsible for approximately 59% of HCC cases in developing countries. Screening of donated blood for transfusion-transmissible infections represents one of the most important strategies for blood-transfusion safety and availability. The presence of this type of infection among blood donors is rare. In a central region of Saudi Arabia, El-Hazmi15 reported that between the years 2000 and 2002, a total of 1.5% of blood donors had HBV infection. In the Al-Ahsa area, as detailed by Panhotra et al,16 the study results showed that 1.9% of blood donors tested HBsAg positive, 3.2% tested HBcAb positive, and 10.1% had developed HBsAb and HBcAb. The Al-Turaifi study17 demonstrated a decrease in the percentage of blood donors that have HBsAg, HBcAb, or HBsAb and HBcAb, to 0.67%, 0.9%, and 7.04%, respectively. Our study results revealed a decrease in the percentage of blood donors that have HBsAg only, HBsAg and HBcAb, or HBsAb and HBcAb, to 0.03%, 0.23%, and 6.9%, respectively. Of the 3014 donors we studied, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 donor (0.03%) for HBsAg only, and 264 (8.8%) for anti-HBc, of whom 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% (6/8) of subjects who tested HBsAg positive and nonreactive in 100% (264/264) of subjects who tested positive for anti-HBc/negative for HBsAg (with or without anti-HBs). Among 2742 donors who tested seronegative (HBs Ag, anti-HBc, and anti-HBs), 1 specimen was reactive according to HBV-NAT results. The frequency of HBV DNA in blood donors who tested seronegative (HBs Ag, anti-HBc, and anti-HBs) was low. Several studies18-27 have reported the effectiveness of HBV NAT in reducing the residual risk of transfusion-transmitted HBV infection in HBsAg-negative blood donations in regions with low/high prevalence of HBV. Although many studies have been published on the value of HBV NAT intercepting seronegative units, this study may interest in areas of the world where hepatitis B remains endemic. In the current study, we detected HBV DNA in 0.04% of the blood donors who tested HBsAg negative. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection among blood donors who test negative for anti-HBs. This finding may be explained by the fact that Saudi Arabia had achieved significant cultural and economic development during the past 2 decades. Also, the low frequency of HBV DNA in blood donors who test HBsAg negative blood donors may result from the presence of a national hepatitis B prevention program. We hope that our results may be useful to our colleagues in other countries as they seek new methods to strengthen the safety of their blood supply. LM Acknowledgments We thank the Deanship of Scientific Research, Majma’ah University, Riyadh, Saudi Arabia, for the financial support it provided us to conduct this study. Abbreviations HBV hepatitis B virus HCC hepatocellular carcinoma HBsAg surface antigen of HBV WP window period RTTI relevant transfusion-transmitted infections NAT nucleic acid testing anti-HBc total HBV core antibody CMIA chemiluminescent microparticle immunoassay PCR polymerase chain reaction rt-qPCR real-time quantitative PCR SPSS Statistical Package for Social Sciences WHO World Health Organization NA nonapplicable References 1. Hollinger FB, Liang TJ. Hepatitis B virus. 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Transfusion . 2013; 53( 10 Pt 2): 2449– 2458. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Laboratory MedicineOxford University Press

Published: May 15, 2018

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