Cervical cancer prevention in the post-cytology era mainly relies on primary HR-HPV screening. Current epidemiological findings have indicated an underestimated role of possible High-Risk HPV (pHR-HPV) types in cervical carcinogenicity and urge for increased availability of genotype-specific information. This could be suggestive for expansion of existing HPV genotyping assays with pHR-HPV types. It is determined pHR-HPV67 is rarely prevalent in cervical cancer, notwithstanding, its close relation to only HR-HPV types. Our objective was to optimize and validate a Next-Generation pHR-HPV67 qPCR assay. These qPCR assays were applied in a HPV67 epidemiological case study.
A triple-target HPV DNA qPCR assay was developed. The occurrence of cross-hybridisation with other genotypes within the same α9-species was evaluated by testing the assays with confirmed positive samples. Limit of quantification (LOQ) and limit of detection (LOD) were determined by a dilution series of synthetic gene fragments. Reproducibility and repeatability were performed by testing HPV67 positive samples in duplicate series and over different days. The epidemiological case study comprised 273 samples, gathered in April 2017, enriched with 100 samples of each aberrant cytological category (LSIL, HSIL).
The cross-hybridization assay confirmed high specificity for HPV67 of the different sets. A LOQ concentration of 33.21 copies/uL and 12.67 copies/uL was obtained for the assays targeting HPV67E6, E7 and L1 respectively (analogue results for E6 and E7). A concentration of 33.21 copies/uL (E6), 6.33 copies/uL (E7) and 3.17 copies/uL (L1) was still detectable in 85% of the cases (LOD). Belgian women with HSIL had a HPV67E6, E7 and L1 prevalence of 8.16% (95% CI: 4.19%;15.28%), 8.16% (95% CI: 4.19%;15.28%) and 7.14% (95% CI: 3.50%;14.01%), respectively (no significant differences: p>0.05, Chi Square test). In LSILs, a HPV67E6, E7 and L1 prevalence of 12.04% (95% CI: 7.17%;19.51%), 12.96% (95% CI: 7.88%;20.59%) and 9.26% (95% CI: 5.11%;16.21%) respectively (p>0.05, Chi Square Test) was determined. Within the screening population, a HPV67E6, E7 and L1 prevalence of 2.22% (95% CI: 1.01%;4.71%), 1.83% (95% CI: 0.78%;4.21%) and 1.83% (95% CI: 0.78%;4.21%) was obtained (p>0.05, Chi Square Test).
Slightly divergent but not significant differences in HPV67L1 prevalence were observed when compared to the HPV67E6 and E7 prevalence. Possible explanations are a variation in PCR efficiency or alternative integration via the HPV L1 gene. The multiple-targeting aspect of the Next-Generation qPCR assay led to the more exact detection of HPV67 and can contribute to an increase in accuracy of HPV detection.