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

• Published in: Journal of clinical virology 2003-09, Vol.28 (1), p.14-26
• Main Authors: Comanor, Lorraine, Elkin, Claudia, Leung, Kimmy, Krajden, Mel, Kronquist, Kathryn, Nicolas, Keith, Horansky, Evelyn, deMedina, Maria, Kittichai, Promrat, Sablon, Erwin, Ziermann, Rainer, Sherlock, Chris
• Format: Article
• Language: English
• Subjects: Genotype; HCV genotype; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepacivirus - physiology; Hepatitis C - diagnosis; Hepatitis C - virology; Humans; Line probe assay; Molecular Probe Techniques; Nucleic Acid Amplification Techniques - methods; RNA, Viral - analysis; RNA, Viral - genetics; Sensitivity and Specificity; TMA–LiPA; Transcription-mediated amplification; Viral Load
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/S1386-6532(02)00234-2

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.