Genotyping of high-risk anal human papillomavirus (HPV): ion torrent-next generation sequencing vs. linear array

Abstract

BackgroundOur next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections.MethodsWe conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections.ResultsIn total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5–96.2; sensitivity: 93.1%, 95% CI: 83.3–98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1–87.7; sensitivity: 61.1, 95% CI: 43.5–76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4–95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114).ConclusionsThe NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0771-z) contains supplementary material, which is available to authorized users.

An economical Nanopore sequencing assay for human papillomavirus (HPV) genotyping

“…Among the 37 discrepant results, 22 were false negatives by Nanopore and 15 were not detected by LA. For the false negatives by Nanopore, more than half (12/22, 54.55%) were mixed infections, and similar finding was reported by other research groups using HPV consensus primers for NGS-based genotyping [10,11]. Other possible causes of false negatives included (1) low viral load, as evident by Specimen 182, from which HPV 16 was missed by both Nanopore and cobas HPV Test;…”

“…Recent advent of next-generation sequencing (NGS) technologies has facilitated high throughput tools for infectious disease diagnostics and epidemiological research. Several research groups have explored utility of Illumina MiSeq and Ion Torrent platforms for HPV genotyping, with comparable sensitivity to well-established line blot assays and broader detection spectrum [10][11][12]. While the reagent cost is comparable to existing commercial assays for large sample batches, these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space.…”

“…Positive agreement was 75-100% for high-risk HPV 31, 33, 35, 39, 51, 52, 56, 58, 59 and 66, and 20% for HPV 68 (n = 5). For non-high risk HPVs, positive agreement was 37.5-100% forHPV 6,11, 40, 42, 53, 54, 55, 61, 62, 70, 72, 73, 81, 82, 83, 84 and 89. There were 2 non-high risk types with 0% positive agreement (HPV 26 and 71).…”

Cross-hybridization between HPV genotypes in the Linear Array Genotyping Test confirmed by Next-Generation Sequencing

“…A recent publication shows that the clinical performance of the LA test within the VALGENT-3 framework, which is designed for comprehensive comparison and clinical validation of HPV tests, is accurate for primary cervical cancer screening [34]. Nevertheless, another recent genotyping study that compares ion torrent-next generation sequencing vs. linear array to detect anal HPVs in individual clinical specimens shows that the NGS assay detects 10 HPV genotypes that are not among the ones detected in LA test: 30, 32, 43, 44, 74, 86, 87, 90, 91, 114 [35]. Some of these HPVs could be cross-reacting with LA test as we demonstrated in this work.…”

A rigorous exploration of anal HPV genotypes using a next‐generation sequencing (NGS) approach in HIV‐infected men who have sex with men at risk for developing anal cancer

“…Previous studies that compared NGS with other HPV detection methods such as INNO‐LiPA DNA hybridization, Electrochemical DNA Chip, Roche Linear Array HPV genotyping (LA) and multiplex PCR reported that NGS is more sensitive and has the ability to detect multiple infections compared to traditional methods and, therefore, may have the potential for use as an alternative method for HPV genotyping and diagnosis of lesions . However, to our knowledge, our study is the first to investigate the presence of anal HPV genotypes using an NGS‐based approach and report their prevalence as varying % read cut points in HIV+ MSM characterized for anal lesion status, demographic and other relevant risk factors.…”