Algorithmic Approach to High-Throughput Molecular Screening for Alpha Interferon-Resistant Genotypes in Hepatitis C Patients

Abstract

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

Principles and Applications of Methods for DNA-Based Typing of Microbial Organisms

“…The patterns produced for a given genotype in the study by Marshall et al (49) were generally consistent, and the patterns were easily interpreted. In contrast, while a consistent, unique fingerprint was generated for each genotype tested in the study by Sreevatsan et al (70), the fingerprints were considerably more complex and difficult to interpret. Furthermore, the fingerprints for HCV types 1a and 1b differed significantly from the patterns for types 1a and 1b presented by Marshall et al (49).…”

“…However, while the intralaboratory reproducibilities of the banding patterns for each viral type were consistent, the interlaboratory reproducibilities of the same patterns for the viral subtypes were poor. In the two studies that used CFLP for genotyping HCV, the structural fingerprints of HCV type 1a and 1b isolates in the study by Marshall et al (49) showed significant differences from those reported by Sreevatsan et al (70). Both studies presented CFLP fingerprints on a 244-bp fragment derived from the 5Ј noncoding region by PCR amplification with the Amplicor HCV kit (Roche Molecular Diagnostics, Branchburg, N.J.).…”

“…CFLP has been applied to genotypic differentiation of HCV isolates (49,70). In both of these studies, genotype analysis by CFLP was successful in differentiating the strains examined.…”

Modulation of the IL-12/IFN-γ axis by IFN-α therapy for hepatitis C

“…Responders also tended to have higher serum ALT concentrations than nonresponders. More responders than nonresponders had genotype 1b, thought to be associated with lower responses to IFN-␣ therapy [44], although this trend lacked significance. Adverse events and PEG-IFN dose were similar between patient groups.…”

Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis

“…For this reason, the use of this amplicon appears to be preferable when subtype information is needed or when more facile analysis of patterns is required, such as in a high-throughput clinical laboratory setting. Sreevatsan et al describe the use of this amplicon for CFLP analysis of a large number of clinical samples of diverse genotypes (21).…”