Successful HCV genotyping of previously failed and low viral load specimens using an HCV RNA qualitative assay based on transcription-mediated amplification in conjunction with the line probe assay.

Background: Hepatitis C virus (HCV) genotyping is a critical part of the diagnostic work-up for chronic hepatitis C. The VERSANT HCV line probe assay (LiPA) marketed by Bayer Corporation requires PCR-derived amplicons for genotyping usually obtained from commercial assays, including Amplicor HCV 2.0 (Amplicor 2.0), Amplicor HCV Monitor 2.0, or SuperQuant. Occasionally, PCR-based methods in conjunction with LiPA fail to give a genotyping result. Although most genotyping failures occur among low viral load specimens, some occur in specimens with relatively high viral loads. The Bayer HCV RNA Qualitative assay (HCV TMA), with a limit of detection of approximately 5-10 IU/ml, is more sensitive than other commercial assays.

Objectives: An HCV genotyping protocol using HCV TMA linked with LiPA (TMA-LiPA) was developed and tested for ability to genotype samples that had previously failed genotyping by PCR-based methods in conjunction with LiPA.

Study Design: Clinical specimens were obtained from eight independent laboratories in Canada and the US and tested with TMA-LiPA at the Bayer Reference Testing Laboratory. Specimens included those that failed to produce a genotype result when a PCR-based assay was used in conjunction with LiPA and specimens for which genotyping was not attempted because the viral load was below the validated cut-off determined in the laboratory of origin.

Results And Conclusions: TMA-LiPA successfully genotyped 68 of 75 (90.7%) specimens that had failed genotyping by PCR-based methods used in conjunction with LiPA and 36 of 40 (90.0%) specimens that were rejected for genotyping due to low viral load. Moreover, TMA-LiPA assigned subtype for 79 of 107 (73.8%) specimens. Our TMA-LiPA results reflected the distribution of HCV genotypes found in North America, and were 100% concordant with those of Amplicor 2.0 in conjunction with LiPA for control specimens genotyped by both assays. TMA-LiPA may prove useful both in optimizing LiPA performance and genotyping patient specimens.