title: Comparison of seven hepatitis B virus (HBV) nucleic acid testing assays in selected samples with discrepant HBV marker results from United States blood donors.
abstract: 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 Poisson distribution impacts results, distinguishing true-positive from false-positive results is challenging. Algorithms are needed to confirm such low-level HBV DNA-positive samples.
  
  
  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 NATs: Ultrio, Ultrio Plus, MPX, and a quantitative assay (SuperQuant). To further explore discrepancies, three additional in-house NATs 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 NATs were as follows: 39 of 107 (36%) with SuperQuant, 40 (30%) with Ultrio, 100 (74%) with Ultrio Plus, and 102 (76%) with MPX. Of the seven NATs, 109 (81%) samples were reactive by at least two assays and thus considered confirmed positive of which 67 (50%) generated a sequence. Ultrio Plus 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 Genotype 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 Genotype B (32%) and E (24%), the latter in an apparent cluster.
  
  
  CONCLUSIONS
  Highly sensitive NATs 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 United States.
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Florence
authors.initials: F
authors.lastname: Enjalbert
authors.forename: David E
authors.initials: DE
authors.lastname: Krysztof
authors.forename: Daniel
authors.initials: D
authors.lastname: Candotti
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Allain
authors.forename: Susan L
authors.initials: SL
authors.lastname: Stramer
mesh_terms: D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000097:blood; D001782:Blood Donors; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood; D005260:Female; D006509:Hepatitis B / Q000097:blood* / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008403:Mass Screening / Q000379:methods; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D014481:United States; D055815:Young Adult
chemical_list: D015415:Biomarkers; D004279:DNA, Viral