Journal of Medical Virology 81:815–825 (2009) Molecular Characterization of Occult Hepatitis B Cases in Greek Blood Donors Antigoni Katsoulidou, 1 Dimitrios Paraskevis, 1 Emmanouil Magiorkinis, 1 Zissis Moschidis, 1 Catherine Haida, 1 Eleni Hatzitheodorou, 1 Agoritsa Varaklioti, 2 Anastasia Karafoulidou, 2 Maria Hatzitaki, 3 Lilian Kavallierou, 4 Athanasia Mouzaki, 5 Evaggelia Andrioti, 6 Chrysanthi Veneti, 7 Athanasia Kaperoni, 8 Eleftheria Zervou, 9 Constantina Politis, 10 and Angelos Hatzakis 1 * ,{ 1 Department of Hygiene and Epidemiology, Athens University Medical School, Athens, Greece 2 2nd Regional Blood Transfusion Center, ‘‘Laiko’’ General Hospital, Athens, Greece 3 Blood Donation Center of Thessaly, Larisa, Greece 4 ‘‘A. Fleming’’ Blood Transfusion Center, Athens, Greece 5 Division of Hematology, Department of Internal Medicine, Medical School University of Patras, Patras, Greece 6 ‘‘Ippokration’’ General Hospital, Athens, Greece 7 ‘‘Konstantopoulio’’ General Hospital N. Ionia, Athens, Greece 8 ‘‘Elpis’’ General Hospital, Athens, Greece 9 Blood Bank, University Hospital, Ioannina, Greece 10 3rd Regional Blood Transfusion Center, General Athens Hospital ‘‘G. Gennimatas,’’ Athens, Greece The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(þ) cases among HBsAg() donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infec- tion in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV-1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(þ)/HBsAg() samples were tested further for HBV serological markers and HBV DNA was quantified by real- time PCR. Molecular characterization was per- formed by sequencing the envelope and poly- merase genes of HBV. Preliminary screening revealed 21 occult cases with the following pat- terns: anti-HBc only: 7 donors, anti-HBc/anti-HBs: 7 donors, anti-HBc/anti-HBe: 5 donors, anti-HBc/ anti-HBs/anti-HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within geno- type D) revealing several amino acid substitu- tions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively. J. Med. Virol. 81:815 – 825, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: blood donors; nucleic acid test- ing; real-time PCR; sequence analysis; phylogeny; a-deter- minant INTRODUCTION Human hepatitis B virus (HBV, family Hepadnavi- ridae, genus Orthohednavirus, species hepatitis B virus) infection is the primary cause of cirrhosis and hepatocellular carcinoma and one of the major causes of death globally. After HBV infection, several distinct outcomes have been defined. Studies based on sero- logical assays have led to the concept that HBV is eliminated after the resolution of an acute infection, although a chronic carrier state may occur in about 5% of adults and 40–80% of neonates [Lee, 1997; Mahoney, 1999; Lok, 2002]. However, the introduction of very sensitive HBV DNA detection tests has revealed the existence of a new entity of HBV infection called ‘‘occult’’ { Chief of National Retrovirus Reference Center. Grant sponsor: Hellenic Scientific Society (Study of AIDS and Sexually Transmitted Diseases, partly supported); Grant sponsor: Safe Blood Biotechnology Suppliers S.A. *Correspondence to: Prof. Angelos Hatzakis, MSc, MD, PhD, Department of Hygiene and Epidemiology, Athens University Medical School, 75 Mikras Asias Street, GR-115 27 Athens (Goudi), Greece. E-mail: ahatzak@med.uoa.gr Accepted 13 January 2009 DOI 10.1002/jmv.21499 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2009 WILEY-LISS, INC.