*3.5. Correlation between HPV Positivity and Clinical Outcome*

Comparing the results obtained from hrHPV testing with the clinical data, we evaluated the overall HPV positivity considering women with abnormal colposcopy findings and women with CIN2+ biopsy. Results for the cervical, vaginal, and urine samples with and without a clinical cut-off are presented in Tables 3 and 4. In Figures 3 and 4 are reported the HPV genotypes' distribution among women with abnormal colposcopy (Figure 3) and among women with CIN2+ lesions (Figure 4).


**Table 3.** HPV-positive women related to colposcopy results.


*Diagnostics* **2022**, *12*, x FOR PEER REVIEW 7 of 11

#### **Table 4.** HPV-positive women related to biopsy results. **Table 4.** HPV-positive women related to biopsy results. **Table 4.** HPV-positive women related to biopsy results.

**Figure 3.** HPV genotypes' distribution among women with abnormal colposcopy findings. **Figure 3.** HPV genotypes' distribution among women with abnormal colposcopy findings. **Figure 3.** HPV genotypes' distribution among women with abnormal colposcopy findings.

**Figure 4.** HPV genotypes' distribution among women with CIN2+ lesions. **Figure 4.** HPV genotypes' distribution among women with CIN2+ lesions.

#### **Figure 4.** HPV genotypes' distribution among women with CIN2+ lesions. **4. Discussion 4. Discussion**

**4. Discussion**  There is presently great interest in the use of self-collected samples as an alternative strategy in cervical cancer screening. The introduction of self-sampling in screening has There is presently great interest in the use of self-collected samples as an alternative strategy in cervical cancer screening. The introduction of self-sampling in screening has There is presently great interest in the use of self-collected samples as an alternative strategy in cervical cancer screening. The introduction of self-sampling in screening has been demonstrated to be more acceptable to women, resulting in improved participation in prevention programs [4,23].

Nowadays, several devices for self-sampling are commercially available, with different performances in sample collection [24–26]. The majority of HPV molecular assays are validated on cervical samples, the gold standard in cervical cancer screening [27]. However HPV assays may give different results when tested on self-taken vaginal and urine samples. Consequently, in order to obtain reproducible results, a specific HPV assay-sample-type validation is necessary [28].

The aim of this study was to evaluate accuracy of the BD Onclarity™ HPV assay on self-collected vaginal samples using a FLOQSwab® and first-void urine using the Colli-Pee® 20 mL device.

The results of the study showed a good overall positive agreement between selfcollected specimens and clinician-collected samples, confirming data already reported in the expanded meta-analysis by Arbyn et al. [6,7]. Recently, a new meta-analysis of test agreement between HPV tests using self-taken vs. clinician-collected samples based on 26 studies (10,071 participants) was published, updating a previous meta-analysis on test accuracy for cervical precancers [29].

The validity of urine samples has been widely debated. On the one hand, this type of sample is easy to collect, overcomes some cultural barriers, and could be useful for surveillance in young populations [30]; on the other hand, the DNA in first-void urine is not always appropriately preserved, thus not allowing the obtaining of an adequate quantity of nucleic acids for HPV analysis [31]. Moreover, urine may collect cells from other nearby anatomical sites, accounting for the higher HPV detection rates and/or multiple infections when compared to cervical samples.

In this study, results from urine samples collected using the Colli-Pee® 20 mL are comparable to results for cervical and vaginal samples, confirming—as previously reported in the literature—the validity of this device for the collection and storage of first-void urine using a preservative medium [15–18,32].

A limitation of the study is that the results come from samples collected from a population of women referred to colposcopy and not from a primary screening setting. However, the purpose of the study was to understand whether the BD Onclarity™ HPV assay could be used in combination with self-samples; hence, it was necessary to have a sufficient number of HPV-positive women to answer this question. For the same reason, previous studies aiming to evaluate HPV testing on clinician-collected vs. self-collected samples have been conducted in a colposcopy setting [18,33,34]. The other main limitation of this study is the small sample size, with participating women being enrolled at only one colposcopy centre. This analysis should therefore be considered a pilot study, and the data obtained could represent the starting point for further larger validation studies on new selfcollection devices paired with the BD Onclarity™ HPV assay. Previous published reports also showed similar limitations regarding sample size as well as a lack of standardized preanalytical methods [14,20,32].

In the present study, vaginal swabs resuspended in 5.5 mL of PreservCyt and 20 mL of first-void urine samples were processed using the same preanalytical protocol as cervical samples by placing 0.5 mL of each sample's starting volume in tubes containing 1.7 mL of BD preserve fluid. Differences in collection procedures, preanalytical procedures, and the nature of the investigated samples may influence HPV detection rates on self-collected samples as compared to cervical specimens, on which the BD Onclarity™ HPV assay protocol and clinical cut-offs had been previously validated. For this reason, we have analysed the data obtained both with the cut-offs set by the manufacturer for cervical samples and without the pre-set cut-offs, still obtaining comparable results in terms of concordance and agreement. However, the different results obtained underline the importance of considering using different testing protocols and analytical cut-offs based on the sample type.

No invalid samples were observed in this pilot study after retesting, irrespective of the sample type. The good sample adequacy observed for vaginal and first-void urine samples may have resulted from collection at the point of care and to reduced time from collection to laboratory testing.

HPV genotyping in self-collected samples represents another strength of the BD Onclarity™ HPV assay, as genotyping could be a good strategy for the triage of HR-HPVpositive women in order to identify those at greater risk of cervical cancer progression, such as HPV 16 and/or 18 positive women. The use of HPV genotyping assays could be very important, particularly for screening programs based on self-sampling due to the impossibility to performing cytology triage on the same sample. Moreover, HPV genotyping on self-taken samples could be helpful to look for persistence of the same HPV genotype at follow-up and as test-of-cure, without the need for a clinician-collected sample at each visit. The possibility of performing a reliable test to verify the success of surgical treatment or the risk of relapse on a self-taken sample can represent another great advantage for women to consider.

Due to the low number of positive samples, we did not perform a statistical analysis of specimens' agreement considering specific HPV genotypes. However, these preliminary results showed a good concordance, especially for HPV 16, even if a further study enrolling a larger number of patients is necessary to obtain statistically significant results.

Up to now, the importance of self-sampling has been stressed to increase adherence to cervical cancer screening programs for women not participating due to socio-cultural or physical barriers. The SARS-CoV-2 pandemic has added another valuable reason to enhance the use of self-sample tests collected at home by women, thus avoiding the need to go to hospital and the risk of COVID exposure and infection [35].
