Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.
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.
Sreevatsan
S
S
Center for Innovative Technologies, ClinCyte, San Diego, California 92121, USA.
Bookout
J B
JB
Ringpis
F M
FM
Pottathil
M R
MR
Marshall
D J
DJ
De Arruda
M
M
Murvine
C
C
Fors
L
L
Pottathil
R M
RM
Barathur
R R
RR
D000465:Algorithms; D003365:Costs and Cost Analysis; D016172:DNA Fingerprinting; D004352:Drug Resistance, Microbial / Q000235:genetics; D004720:Endonucleases / Q000378:metabolism; D005821:Genetic Techniques / Q000191:economics; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000453:epidemiology / Q000821:virology*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D016898:Interferon-alpha; D004720:Endonucleases