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Additional file 1: Table S1. Intralaboratory reproducibility of HPV Selfy according to Meijer’s guidelines. HPV Selfy assay was performed twice on a subpopulation of 521 samples of which 157 samples positive to HC2. Overall concordance observed was 94.6% (kappa value of 0.87). Table S2. Interlaboratory reproducibility of HPV Selfy according to Meijer’s guidelines. HPV Selfy assay was performed in another laboratory (MediChrom) on a subpopulation of 500 samples of which 150 samples positive to HC2. Overall concordance observed was 93.6% (kappa value of 0.85). Table S3. Intra-laboratory reproducibility of genotype findings of HPV Selfy. Data are presented as number of each genotype detected in each run (i.e. run1 and/or run 2), and numbers do not count up to the total number of HR-HPV positive samples due to multiple infections.

HPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.97 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population. Clinical validation of HPV Selfy on self‑collected vaginal samples (VALHUDES) Next, we aimed at evaluating HPV Selfy performance on self-collected samples, as indicated by VALHUDES protocol. Hence, we needed to assess whether HPV testing on vaginal self-samples was as accurate as HPV testing on a cervical sample taken by a clinician. To do so, we identified 119 CIN2+ cases (age 25–65 years) and 791 ≤ CIN1 cases, for which we had available paired cervical specimen and self-collected vaginal samples. HPV Selfy testing in self-collected samples was found similarly sensitive (93/96; relative sensitivity 0.97; 95% CI 0.90–1.04) and specific (722/745; relative specificity 0.97; 95% CI 0.95–0.99) to detect CIN2+ in the total study population (Table 4), in comparison with HPV Selfy performed on paired ThinPrep. Thus, HPV Selfy assay fulfills VALHUDES requirements for use of HR-HPV DNA tests on self-collected samples according to non-inferiority analysis (relative sensitivity > 0.90 with T = 1.70, p = 0.044; relative specificity > 0.95 with T = 1.87, p =0.031). Secondary objectives of VALHUDES protocol include the assessment of the absolute accuracy of HR-HPV DNA test applied according to the sampling device and the proportion of adequate samples as determined by amplification of an internal control (a ubiquitous human gene). HPV Selfy assay provides a human beta-globin internal control, used to evaluate sample quality. In the whole study cohort, mean Ct value for the human beta-globin internal control for the HPV Selfy test on self-collected samples, obtained with the direct analysis on self-collected samples, was 26.1 Ct (median value 25.9 Ct, maximum 30.7 Ct, minimum 16.5 Ct). In the subgroup of women with biopsy-diagnosis of cervical lesions CIN2+, the same analysis resulted in 26.7 Ct (median value 26.5 Ct, maximum 30.7 Ct, minimum 24.2 Ct). This means that all women were able to self-collect a similar amount of specimen, confirming self-collected samples’ quality adequacy and so the easiness of the self-sampling procedure using the FLOQSwabs® (Copan, Brescia, Italy).

Background According to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed – Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples. Methods For each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO—National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture®2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test. Results HPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population. Conclusions HPV Selfy fulfills all the requirements of the international Meijer’s guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling. Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/2018