Detection of diagnosis escape variants of Hepatitis B virus by in house polymerase chain reaction assay

Abstract

P. Balkumar Reddy, R. M. Mukherjee, J. Aparna, Sasikala Mitnala, P. N. Rao, R. Gupta and D. N. Reddy

Quantitative detection of Hepatitis B virus (HBV) DNA in serum by real time polymerase chain reaction (PCR) assay emerged as a gold standard in guided anti viral therapy. HBV, due to its quasispecies nature, whether evade detection by the commercially used diagnostic PCR assays have not been elucidated so far. In this study, an in house nested PCR assay employing mismatched primer was found to detect HBV variants that escape detection by a commercial real time (Cobas TaqMan HBV test) PCR assay. Sera of 178 HBsAg +ve subjects were screened for HBV DNA by both the assays and sequence verified. The present assay detected 80% of 108 real time PCR positive subjects of which, 79.7% had single band (858bp) of HBV DNA and 20.3% showed mixed type (858 + 192 bp). HBV specific192 bp amplicon containing A1762T and G1764A mutations has also been detected in 5 (7.1%) out of 70 real time PCR negative subjects. The data provided important information on persistent heterogeneity of HBV in single infected individual that escape routine detection by the commercial assay, and, thus, might have implication on the estimation of viral load as well as treatment outcome in HBV infection.