N: number; CI: 95% confidence interval; CIN: cervical intraepithelial neoplasia
+/+ positive on FVU and cervical samples, +/- positive only on cervical samples, -/+ positive only on FVU samples, -/- negative on both sample types.
Note: Color legend: for the concordance: dark green (1.00 ≥ K > 0.80): excellent; light green (0.80 ≥ K > 0.60): good; yellow (0.60 ≥ K > 0.40): moderate; orange (0.40 ≥ K > 0.20): fair; red (0.20 ≥ K > 0.00): poor.
Discussion
The introduction of self-sampling in cervical cancer screening programs, further enhanced by the COVID19 pandemics (17), is an important instrument to reach 70% screening coverage as proposed in the WHO call to action (1). The similar clinical accuracy of PCR-based HPV tests on self-samples and clinician-collected cervical scrapes has been demonstrated in previous validation studies for other assays (10–15).
The present study demonstrated that the use of OncoPredict HPV SCR assay on self-collected vaginal specimens FLOQSwabs and resuspended in 5 ml of eNat and FVU collected using Colli-Pee FV5000 has a similar clinical accuracy to detect ≥CIN2 and CIN3 lesions as compared to clinician-collected cervical samples. Clinical sensitivity of OncoPredict HPV SCR assay on FVU and vaginal samples was similar to cervical specimens, however specificity on vaginal samples was lower when applying manufacturer cut-off values. Cut-off optimization on vaginal self-collected samples resulted in an improvement in specificity without compromising sensitivity. A lower specificity for the detection of ≥CIN2 as compared to cervical specimen was also reported for the validation of BD Onclarity HPV test on FLOQSwabs resuspended in 3 ml of BD HPV self-collection diluent (18). On the other hand, in the Belgian VALHUDES, where vaginal samples were resuspended in 20 ml of PreservCyt, a posteriori cut-off determination was necessary to improve the clinical sensitivity, but not for the specificity (11,12).
Different preanalytical workflows of self-collected vaginal samples may affect on clinical accuracy of the test. Therefore, optimization and standardization of the procedures for handling and testing of self-samples are fundamental to ensure an optimal performance of the assay (19). Presently, the VALHUDES protocol has been developed to assess the performance of HPV tests in combination with self-collection devices (9). In the European VALHUDES, vaginal swabs have been collected using FLOQSwabs resuspended in 5 ml of eNat, while FVU was collected using a 20 ml Colli-Pee device. eNat is a transport medium that allows the preservation of nucleic acids, denaturation of proteins and inactivation of microbial agents. It has been previously used in combination with HPV molecular assays (20,21);two studies demonstrated a good analytical performance of FLOQSwabs resuspended in 5 ml of eNat as compared to cervical samples (22,23). Moderate to excellent agreement between vaginal and cervical specimens was also demonstrated in the present study.
Both urine and vaginal self-collected samples are well accepted by women (24), in particular this study confirmed that FVU is a non-invasive collection method with clinical accuracy for ≥CIN2 lesions comparable to cervical specimens, as previously reported (14,15).
Ensuring sample adequacy is crucial, particularly for self-collection, in order to prevent false-negative results (25–27). One of the main advantages of OncoPredict HPV SCR assay is the inclusion of a thorough quality assessment, both for the preanalytical and analytical phases. The assay allows to determine the efficiency of nucleic acid extraction and potential PCR inhibition through the use of external calibrators, as well as assessing adequacy of sample collection through a quantitative cellularity assessment. In most molecular assays an internal housekeeping gene is used for both sample adequacy and amplification assessment. Recent studies have underlined the importance of identifying the cause of invalidity (25) of performing quality controls for the assay in a separate reaction well (26). In the present study, no invalid result related to PCR inhibition was detected, underlying the good performance of the analytical process. The invalidity due to a low extraction efficiency could be attributed to errors in specific nucleic acid extraction runs that may be resolved by retesting samples after a new extraction. On the contrary, in case of low cellularity samples, in absence of other invalidity reasons, sample collection should be repeated (25). In general, the invalidity rate was higher in cervical samples than in self-collected samples. This could be related to different limits of acceptable cellularity for cervical and self-collected samples. Moreover, as previously discussed, in the present study vaginal samples were resuspended in 5 ml of eNat while cervical swabs in 20 ml of PreservCyt which may have resulted in lower sample cellularity. Finally, the inclusion of the QC module in OncoPredict HPV SCR assay allows to accurately verify the sample adequacy, avoiding false negative results in cervical cancer screening.
OncoPredict HPV SCR assay is a limited genotyping assay, identifying HPV16, HPV18 and/or “other” hrHPV genotypes, whereas OncoPredict HPV Quantitative Typing (QT) is a full genotyping assay that can distinguish all the 12 hrHPV types separately. Both assays have been independently validated on cervical and self-samples withing VALGENT and VALHUDES frameworks (Latsuzbaia et al., submitted, 16,28), respectively. hrHPV-positive specimens identified with OncoPredict HPV SCR assay may benefit from complete genotyping using QT assay as a reflex test.
In conclusion, following a posteriori cut-offs adjustment the OncoPredict HPV SCR assay demonstrated similar clinical accuracy for ≥CIN2 lesions on self-collected vaginal and FVU samples compared to testing on clinician-collected cervical samples.