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BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI). STUDY DESIGN AND METHODS: From January 2005 to December 2006, a total of 383,267 blood units were screened for hepatitis B surface antigen (HBsAg) and HBV DNA in two transfusion centers in Madrid, using either individual-donation nucleic acid testing (ID-NAT) or minipool (MP-NAT) of eight donations (MP8). Samples positive for HBV DNA and negative for HBsAg were confirmed by a second molecular test, the viral DNA was quantified, and a genome fragment including the region encoding the major hydrophilic region (MHR) of HBsAg was sequenced. RESULTS: The overall yield of HBV DNA–positive, HBsAg-negative units was 1 in 21,282 (18 cases), higher when using ID-NAT than MP8-NAT (1:9862 vs. 1:51,011; p < 0.01). Four donations (1/95,817) were collected during the infectious pre-HBsAg WP, one during an early recovery stage, and the remaining 13 (1/29,482) were OBIs, six of whom had no detectable antibody to HBsAg. Low-level Genotype D HBV DNA was detected in all OBI cases; the frequencies of this genotype and MHR amino acid substitutions were significantly higher than reported from unselected Spanish HBsAg carriers. Donors with OBI had normal aminotransferase levels and were significantly older than donors carrying HBsAg. CONCLUSIONS: Blood donors in the WP and with OBI are not uncommon in Madrid and are detected at a higher frequency with ID-NAT than MP-NAT. [ABSTRACT FROM AUTHOR]