Dataset Information

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.

SUBMITTER: Vernon SD

PROVIDER: S-EPMC86168 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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

Vernon S D SD Unger E R ER Williams D D

Journal of clinical microbiology 20000201 2

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 ...[more]

PMID: 10655362

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Project description:Hybrid Capture 2 (hc2), a clinical test for carcinogenic human papillomavirus (HPV) DNA, has proven to be a sensitive but only modestly specific predictor of cervical precancer and cancer risk. Some of its nonspecificity for clinical end points can be ascribed to cross-reactivity with noncarcinogenic HPV genotypes. However, the reference genotyping tests that have been used for these comparisons are also imperfect. We therefore sought to describe further the HPV genotype specificity of hc2 by comparing the hc2 results to paired results from two related PGMY09/11 L1 primer-based HPV genotyping assays: Linear Array (LA) and its prototype predecessor, the line blot assay (LBA). LA and LBA results were considered separately and combined (detection by either assay or both assays) for 37 individual HPV genotypes and HPV risk group categories (carcinogenic HPV > noncarcinogenic HPV > negative). Baseline specimens from 3,179 of 3,488 (91.5%) women referred to ALTS (a clinical trial to evaluate the management strategies for women with atypical squamous cells of undetermined significance [ASCUS] or low-grade squamous intraepithelial lesions) because of an ASCUS Papanicolaou smear were tested by all three assays. Among single-genotype infections with genotypes targeted by hc2 as detected by either PCR assay, HPV genotype 35 (HPV35) (86.4%), HPV56 (84.2%), and HPV58 (76.9%) were the most likely to test positive by hc2. Among single-genotype infections with genotypes not targeted by hc2 as detected by either assay, HPV82 (80.0%), HPV66 (60.0%) (recently classified as carcinogenic), HPV70 (59.1%), and HPV67 (56.3%) were the most likely to test positive by hc2. Among women who tested negative for carcinogenic HPV by both PCR tests and were positive for noncarcinogenic HPV by either test, 28% of women were hc2 positive. Conversely, 7.8% of all hc2-positive results in this population were due to cross-reactivity of hc2 with untargeted, noncarcinogenic HPV genotypes. In conclusion, hc2 cross-reacts with certain untargeted, noncarcinogenic HPV genotypes that are phylogenetically related to the targeted genotypes, but the degree of cross-reactivity may be less than previously reported.

Project description:ImportanceHigh-risk human papillomavirus (hrHPV) persistent infection is the major etiology of cervical precancer and cancer. Noninvasive self-sampling HPV testing is a promising alternative cervical cancer screening for avoiding stigma and improving patient willingness to participate.ObjectiveTo investigate the feasibility and accuracy of menstrual blood (MB) hrHPV capture sequencing in hrHPV detection.Design, setting, and participantsThis cohort study collected 137 sanitary pads from 120 women who were premenopausal and had hrHPV as detected by cervical HPV GenoArray testing. Patients were recruited from September 1, 2020, to April 1, 2021, at Central Hospital of Wuhan, China. Target capture sequencing was performed to determine hrHPV genotypes in MB. Sanger sequencing was performed as the criterion standard for detecting hrHPV genotypes among enrolled women. Data were analyzed from April 1 through June 1, 2021.Main outcomes and measuresComplete concordance, incomplete concordance, and discordance of MB hrHPV capture sequencing and conventional HPV testing were defined according to genotype overlapping levels. Concordance of the 2 detection methods and comparative power of MB hrHPV capture sequencing during different menstrual cycle days (MCDs) were the main outcomes.ResultsA total of 120 enrolled women with hrHPV (mean [SD; range] age, 33.9 [6.9; 20.0 -52.0] years) provided 137 sanitary pads. The overall concordance rate of MB hrHPV capture sequencing and cervical HPV testing was 92.7% (95% CI, 88.3%-97.1%), with a κ value of 0.763 (P < .001). Among 24 samples with incomplete concordance or discordant results, 11 samples with additional hrHPV genotypes (45.8%), 5 true-negative samples (20.8%), and the correct hrHPV genotypes of 2 samples (8.3%) were correctly identified by MB hrHPV capture sequencing. MB hrHPV detection of hrHPV was equivalent on different MCDs, with an MB hrHPV-positive rate of 27 of 28 patients (96.4%) for MCD 1, 52 of 57 patients (91.2%) for MCD 2, 27 of 28 patients for MCD 3, 4 of 4 patients (100%) for MCD 4, and 3 of 3 patients (100%) for MCD 5 (P = .76). The sensitivity of the MB hrHPV capture sequencing was 97.7% (95% CI, 95.0%-100%).Conclusions and relevanceThese findings suggest that MB hrHPV capture sequencing is a feasible and accurate self-collected approach for cervical cancer screening. This study found that this method is associated with superior performance in identification of HPV genotypes and true-negative events compared with cervical HPV testing.

Project description:BackgroundWe recently described a method for unbiased detection of all known human papillomaviruses (HPV) types with the potential for the determination of their variant and integration from the resulting whole genome sequence data. Considering the complex workflow for target-enriched next generation sequencing (NGS), we focused on the reproducibility and limit of detection (LOD) of this new universal HPV typing assay in this study.ResultsWe evaluated the reproducibility and LOD for HPV genotyping based on our recently published method that used RNA-baits targeting whole genomes of 191 HPV types, Agilent SureSelect protocol for target enrichment and Illumina HiSeq 2500 for sequencing (eWGS, enriched whole genome sequencing). Two libraries, prepared from pooled plasmids representing 9 vaccine HPV types at varying input (1-625 copies/reaction), were sequenced twice giving four replicates for evaluating reproducibility and LOD. eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R2 = 1) and between experiments (R2 = 0.99). The number of mapped reads was positively correlated to copy number (β = 13.9, p < 0.0001). The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample. HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present. eWGS showed no bias for HPV genotyping under single or multiple infection (p = 0.16-0.99).ConclusionsThe universal eWGS method for HPV genotyping has sensitivity, competitive with widely used consensus PCR methods with reduced type competition, and with the potential for determination of variant and integration status. The protocol used in this study, using defined samples varying in complexity and copy number, analyzed in replicate and duplicate assays, is applicable to most WGS methods.

Dataset Information

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

Publications

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

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