S. Sreevatsan et al., ALGORITHMIC APPROACH TO HIGH-THROUGHPUT MOLECULAR SCREENING FOR ALPHA-INTERFERON-RESISTANT GENOTYPES IN HEPATITIS-C PATIENTS, Journal of clinical microbiology, 36(7), 1998, pp. 1895-1901
This study was designed to analyze the feasibility and validity of usi
ng Cleavase Fragment Length Polymorphism (CFLP) analysis as an alterna
tive to DNA sequencing for high-throughput screening of hepatitis C vi
rus (HCV) genotypes in a high-volume molecular pathology laboratory se
tting. By using a 244-bp amplicon from the 5' untranslated region of t
he HCV genome, 61 clinical samples received for HCV reverse transcript
ion-PCR (RT-PCR) were genotyped by this method. The genotype frequenci
es assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b) 1
3.1% for type 2b, and 5% type 3a. The results obtained by nucleotide s
equence 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 ge
notype frequencies also concurred with the national estimates (N. N. Z
ein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of
these samples in parallel in a different research laboratory reproduce
d 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 c
ost analysis for genotyping of HCV by line probe assay, CFLP analysis,
and automated DNA sequencing indicated that the average cost per ampl
icon was lowest for CFLP analysis, at $20 (direct costs). On the basis
of these findings we propose that CFLP analysis is a robust, sensitiv
e, specific, and an economical method for large-scale screening of HCV
-infected patients for alpha interferon-resistant HCV genotypes. The p
aper describes an algorithm that uses as a reflex test the RT-PCR-base
d qualitative screening of samples for HCV detection and also addresse
s genotypes that are ambiguous.