• anti-HBe = antibody to HBeAg;
  • anti-HBs = antibody to HBsAg;
  • C = sequences of HBV core genes;
  • CID50 = chimpanzee infectious dose;
  • HBeAg = hepatitis B e antigen;
  • HN-PCR = heminested PCR;
  • IAC = immuno-affinity capture;
  • P = sequences of HBV polymerase genes;
  • QSG = Qiagen silica gel;
  • QSG-60 = QSG method wherein the samples are heated with lysis buffer at 60°C for 10 minutes;
  • QSG-PK = QSG method wherein the samples are heated at 58°C for 60 minutes with proteinase-K;
  • S = sequences of HBV surface genes;
  • UCSF = University of California, San Francisco

BACKGROUND: Occult viremia occurring before the appearance of HBsAg or after the disappearance of HBsAg is detectable by gene amplification technologies whose efficiency depends on nucleic acid preparation.

STUDY DESIGN AND METHODS: To isolate HBV DNA from viremic plasma, immunoaffinity capture (IAC) of intact HBV with biotinylated pre-S1 antibodies coupled to streptavidin-coated magnetic beads was evaluated. IAC was compared with a silica-gel method (Qiagen [QSG]) and its two modifications wherein the samples were heated with lysis buffer at 60oC for 10 minutes (QSG-60) or at 58°C for 60 minutes with proteinase-K (QSG-PK). Each HBV DNA sample was tested by heminested PCR amplification of the HBV gene sequences. A total of 36 coded serum samples were tested, including three HBsAg-positive controls and 33 former chronic HBV carriers who had seroconverted (developed antibody to HBsAg [anti-HBs]). Commercially available seroconversion panels (PHM 907, 911, and 922) were similarly tested for window-period viremia.

RESULTS: In the 33 former chronic HBV carriers who had seroconverted, IAC revealed HBV DNA in 17 samples, whereas it was revealed in only 11 samples by QSG-PK (p = 0.031), 10 by QSG-60 (p = 0.016), and 9 by QSG (p = 0.0078). However, HBV DNA was not amplified from the 17 samples at 1-in-10 dilutions; thus, they were considered to have low-level viremia. IAC revealed HBV DNA as early as or earlier than the other methods in PHM 907, 911, and 922 panels.

CONCLUSION: IAC is apparently an optimal method of sample preparation for amplification of HBV DNA in patients in the pre-HBsAg window period, and for detecting low-level viremia persistent in several individuals who were former chronic HBV carriers who had seroconverted (developed anti-HBs).