Christophe Pasquier

HPV genotyping in clinical samples using long‐read single‐molecule real‐time sequencing

AbstractHuman papillomavirus (HPV) genotyping is widely used, particularly in combination with high‐risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real‐time sequencing (SMRT) and evaluated its performance against MY09‐11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09‐11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR‐HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR‐HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Human papillomavirus testing using HPV APTIMA® assay and an external cellularity control in self‐collected samples

AbstractIn cervical cancer screening programs, the detection of high‐risk human papillomavirus (HR‐HPV) is now widely implemented on physician‐collected samples and has expanded to include self‐collected samples. The use of a cellularity control (CC) is needed to reduce false‐negative HPV results. An external mRNA CC for the HPV APTIMA® assay was assessed for its analytical performance and the results were compared with both cervix cytobrush samples taken by physicians and self‐collected vaginal samples from 148 women. The performance of the CC was adjusted to control for the presence of cellular mRNA in the ThinPrep® and Multitest® transport media. This CC is user‐friendly but implies to perform two independent assays on PANTHER® automate. Self‐collected vaginal sampling gives a lower median CC results (13.2 vs. 16.9 min) but a higher risk of negative CC results (3.3 vs. 0%). The usefulness of the CC for the HR‐HPV assay may be optimized by the definition of a threshold for a minimum cell number to be tested to increase confidence in HPV‐negative results. The systematic use of an RNA CC increases confidence for HPV RNA assays on self‐collected vaginal samples.