Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture

Abstract

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (P = 0.001) and to detect an HPV type (P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.

FIG. 1

Comparison of positive and negative results by the Hybrid Capture assay and line blot assay for samples infected with different HPV types. The Hybrid Capture assay probe A cocktail contains HPV types 6, 11, 42, 43, and 44 and the probe B cocktail contains HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56. For a sample to have concordant results by both assays, the type detected (or not detected) by the line blot assay had to be the one included in the probe cocktail and vice versa. All other samples were considered to have discrepant results. The HPV types that may account for the Hybrid Capture assay-positive, line blot assay-negative samples with discrepant results are shown.