Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA.

Nucleic acid amplification technique-based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

Evaluation of 10 commercial diagnostic kits for in vitro expressed hepatitis B virus (HBV) surface antigens encoded by HBV of genotypes A to H.

Genetic variability of the hepatitis B virus (HBV) constitutes one of the major challenges for diagnosis of HBV infection. It is plausible that amino acid substitutions in the "a" determinant of the HBV surface antigen (HBsAg) that affect antigenic sites, whether originating from genetic diversity or from mutations in the HBV strain itself, will affect the sensitivity of some diagnostic kits. In fact, recent studies have indicated that some diagnostic kits had false negative results with particular HBsAg mutants. There have been, however, few substantial studies evaluating sensitivities of diagnostic kits to the HBsAg encoded by different HBV genotypes. Our recent study found that 10 diagnostic kits available in Japan were able to detect HBsAg irrespective of whether it originated from HBV genotypes A, B or C, with the latter two genotypes being the dominant species in East Asia. In this study, we extended our previous efforts by assessing the ability of diagnostic kits to detect recombinant HBsAg derived from HBV genotypes A to H. Our results demonstrated that 9 out of 10 diagnostic kits evaluated were able to detect as low as 0.2 International Units (IU)/ml HBsAg, irrespective of HBV genotype. The genotypic differences in the HBV family thus appear to have little impact on the sensitivity of currently available HBsAg diagnostic kits.

Reactivity of genotypically distinct hepatitis B virus surface antigens in 10 commercial diagnostic kits available in Japan.

Hepatitis B virus (HBV) surface antigen (HBsAg) is one of the most important serological markers of current HBV infection. However, there are significant antigenic variations of HBsAg caused by genotypic diversity as well as mutation of the HBV genome. It is predictable that amino acid substitutions occurring in the HBsAg "a" determinant of a particular HBV genotype will affect the sensitivity of some diagnostic kits, since all the diagnostic kits currently available utilize monoclonal and/or polyclonal antibodies against the "a" determinant. One possible concern is that there may be a significant variation in the sensitivity of HBsAg diagnostic kits to HBsAg encoded by HBV of different genotypes, which might result in a failure to detect HBsAg of a particular HBV genotype. In this study, we assessed the reactivity of HBsAg specimens derived from three different HBV genotypes (A, B, and C) that are prevalent in Japan by 10 commercially available EIA (enzyme immunoassay), CLIA (chemiluminescent immunoassay), and CLEIA (chemiluminescent enzyme immunoassay) diagnostic kits. Specimens included both clinical samples and recombinant HBsAg. Our results showed that all the diagnostic kits evaluated were able to detect HBsAg irrespective of HBV genotypes. At the same time, it is apparent that some, but not all of the kits showed clear differences in sensitivity to the three HBV genotypes.