Comparison of seven hepatitis B virus ( HBV ) nucleic acid testing assays in selected samples with discrepant HBV marker results from U nited S tates blood donors

Background Sensitive triplex nucleic acid tests (NATs) are implemented for blood donation screening worldwide. Assays have variable ability to detect low‐level hepatitis B virus ( HBV ) DNA . At borderline DNA detection levels, where P oisson distribution impacts results, distinguishing true‐positive from false‐positive results is challenging. Algorithms are needed to confirm such low‐level HBV DNA –positive samples. Study Design and Methods A total of 135 blood donor samples reactive by one or more HBV markers that provided discrepant results were tested undiluted with four commercial NAT s: U ltrio, U ltrio P lus, MPX , and a quantitative assay ( S uper Q uant). To further explore discrepancies, three additional in‐house NAT s including real‐time polymerase chain reaction ( PCR ) and nested PCR and sequencing were performed. Results The numbers reactive of these 135 “difficult” samples by four commercial NAT s were as follows: 39 of 107 (36%) with S uper Q uant, 40 (30%) with U ltrio, 100 (74%) with U ltrio P lus, and 102 (76%) with MPX . Of the seven NAT s, 109 (81%) samples were reactive by at least two assays and thus considered confirmed positive of which 67 (50%) generated a sequence. U ltrio P lus and MPX performed similarly as above (80%‐85% detected of 109 and 81%‐90% of 67, respectively). Older (median, 49 years), HBV core antibody–reactive donors carried predominantly G enotype A (58%) with high‐frequency amino acid substitutions in the major hydrophilic region of the S ‐protein. Younger (median, 24 years) hepatitis B surface antigen–positive donors carried wild‐type strains predominantly G enotype B (32%) and E (24%), the latter in an apparent cluster. Conclusions Highly sensitive NAT s require new confirmatory algorithms as presented optimally using different genomic regions or sequence generation. The introduction of immigration‐related HBV genotypes may impact HBV epidemiology in the U nited S tates.