1. HBV DNA International Standard: At present, there is no international standard of HBV-DNA to evaluate the sensitivity and specificity of the HBV-DNA NAT assays. We participated in a study organized by NIBSC and WHO (with 24 organizations world wide participating) to develop several HBV- DNA candidates of standards for quantitative assays of HBV DNA infections. The collaborative study showed that 3 candidate preparations for the international standards of HBV DNA have a mean estimates of 6.42, 6.30, and 5.03 log10 geq/ml (geq=genome equivalent). These standards will be suggested for use to evaluate the sensitivity of commercial HBV NAT assay products. 2. Silent HBV: The uncommon finding of """"""""silent"""""""" hepatitis B virus (HBV), characterized by the presence of HBV DNA in serum or liver tissue in the absence of hepatitis B surface antigen (HBsAg), anti-HBc and anti-HBs in serum, is thought to be caused by HBV mutants that produce a very low level of HBsAg, which escape detection by currently available serologic tests. This is an issue of blood transfusion safety. HCC and adjacent liver tissues from forty-three patients whose sera were HBsAg-negative, as well as three positive and four negative controls (a total of 87 DNAs) were further studied for HBV DNA using nested PCR, including 9 from various areas of the USA, 22 from Vancouver, Canada, and 12 from China. HBV DNA was detected by PCR in 5 of the 9 patients (55%)from HBsAg-negative patients from the USA, in 12 of the 22 from Canada (55%), and in 12/12 from Qidong, China (100%). Sequencing of the 241-bp S gene PCR product has been completed in 20 patients; each contains the expected S gene sequence with one or more nucleotide variations differing from the consensus sequence and from each other, which rules out the possibility that the positive results could be due to contamination. Genotyping was determined using the type-specific amino acids in the S protein. Among the four silent HBV patients from the USA, two had HBV of genotype A, and two genotype C. Among 5 silent HBVs from Canada, one was genotype B, four were genotype C. Replication of HBV was determined by the presence of replicative intermediates covalently closed circular (CCC) HBV DNA. In HBsAg-positive patients, CCC DNA was found in 3/5 (60%), while in silent HBV, was found in 1/9(11%), in residual HBV, was found in 1/8(12.5%). Thus, in HCC patients from USA and Canada, who are HBsAg-negative, HBV DNA could be detected in 55% by nested PCR. Since """"""""silent"""""""" HBV is defined by its ability to escape detection by all serologic tests for HBsAg, these findings are of interest for blood donor screening, as well as in understanding the development of such complications of chronic HBV infection as HCC.
