Accuracy of self-collected vaginal dry swabs using the Xpert human papillomavirus assay

Background Polymerase chain reaction-based Xpert human papillomavirus (HPV) assay is a rapid test that detects high-risk HPV (hrHPV) infection. This point-of-care test is usually performed by collecting a cervical specimen in a vial of PreservCyt® transport medium. We compared HPV test positivity and accuracy between self-collected sample with a dry swab (s-DRY) versus physician-collected cervical sampling using a broom like brush and immediate immersion in PreservCyt (dr-WET). Methods In this cross-sectional study, we recruited 150 women ≥ 18 years old attending the colposcopy clinic in the University Hospital of Geneva. Each participant first self-collected a vaginal sample using a dry swab and then the physician collected a cervical specimen in PreservCyt. HPV analysis was performed with Xpert. Part of the PreservCyt-collected sample was used for hrHPV detection with the cobas® HPV test. HPV test positivity and performance of the two collection methods was compared. Results HPV positivity was 49.1% for s-DRY, 41.8% for dr-WET and 46.2% for cobas. Good agreement was found between s-DRY and dr-WET samples (kappa±Standard error (SE) = 0.64±0.09,), particularly for low-grade squamous intraepithelial lesions (LSIL+) (kappa±SE = 0.80±0.17). Excellent agreement was found between the two samples for HPV16 detection in general (kappa±SE = 0.91±0.09) and among LSIL+ lesions (kappa±SE = 1.00±0.17). Sensitivities and specificities were, respectively, 84.2% and 47.1%(s-DRY), 73.1% and 58.7%. (dr-WET) and 77.8% and 45.7% (cobas) for CIN2+ detection. The median delay between sampling and HPV analysis was 7 days for the Xpert HPV assay and 19 days for cobas. There were 36 (24.0%) invalid results among s-DRY samples and 4 (2.7%) among dr-WET (p = 0.001). Invalid results happened due to the long interval between collection and analysis. Conclusion Self-collected vaginal dry swabs are a valid alternative to collecting cervical samples in PreservCyt solution for HPV testing with the Xpert HPV assay. Impact HPV self-collection with dry cotton swabs might assist in the implementation of an effective screening strategy in developing countries. Trial registration International Standard Randomized Controlled Trial Number Registry ISRCTN83050913


Recent development of tests for high-risk human papillomavirus (hrHPV) infection has created 2
an important change in our understanding of cervical cancer screening. 3 Overwhelming evidence from several randomized trials have shown that HPV screening is 4 more effective than cytology in preventing cervical cancer. It demonstrates better sensitivity 5 and allows less frequent screening 1, 2 . 6 Besides developed countries, which progressively are incorporating HPV testing in their 7 national cervical cancer-screening program and updating their current guidelines 3 , developing 8 countries, following the recommendations of the World Health Organization, are evaluating the 9 HPV testing as a primary screening tool 4 . 10 HPV testing is associated with decreased cervical cancer-related mortality 5, 6 and it gives the 11 possibility to use a self-vaginal sampling (self-HPV), which is considered as accurate as 12 physician-cervical sampled HPV tests 7 . 13 Not only for low resource settings, but equally for developed countries like Switzerland, the 14 introduction a point-of-care, rapid, non-batch assay facilitating same-day screen and 15 management strategies is essential. This approach would minimize repeat visits, allowing 16 therefore, most of the eligible women to participate in the program. The ideal system should 17 be modular and easily integrated into modest-income settings. 18 Furthermore PCR-based hrHPV tests with a higher analytic sensitivity should be preferred 19 because they ensure similar accuracy between clinician-collected and self-collected samples 7 . 20 Such a point-of-care testing seems to be the Xpert HPV assay (Cepheid, Sunnyvale, CA, 21 USA), a non-batch and qualitative real-time PCR assay for the detection of hrHPV DNA. The 22 assay is formatted in a single-use test cartridge. A single test can be completed in 1 hour, 23 allowing same-day screen-and-treat, which reduces the potential loss to follow-up. The 24 developed system provides a modular flexibility ranging from 1 to 80 test processing modules 25 able to deliver from one to 471 HPV test a day. 26 Until now the Xpert HPV test was evaluated with specimens previously collected into ThinPrep 27 (Hologic) vials containing a methanol-water solution (PreservCyt transport medium) 8 . This 28 approach may be impractical and unavailable, because of flammability, toxicity and cost, 29 especially in low resource settings. In developed countries, women have expressed concerns 30 for liquid transport medium, thinking that the test quality is reduced by accidentally spilling out 31 some of the transport medium 9, 10 . 32 In a previous self-HPV study we found that swabs transported in a dry state were as accurate 33 as those obtained with swabs shipped in a wet transport medium, in terms of quality of 34 results 11 . 35 The possibility of using self-obtained specimens stored at ambient temperature without 36 transport media would clearly enhance and simplify the utility of Self-HPV. Moreover it 37 reduces the costs of the method, which might be attractive for a point-of-care strategy. 38 The objective of our study is to compare performance diagnosis for hr-HPV infection of Self-39 HPV using dry swabs (S-DRY) and Physician collected sample with swab immediately 40 immersed in PreservCyt (dr-WET). The comparison will be carried out after testing with the 41 Xpert HPV. 42 If the S-DRY approach with the Xpert HPV assay is feasible, it will assist the implementation 43 of a cost-effective screening strategy worldwide; in developing countries, by overcoming 44 material and human barriers and minimizing need for repeat visits, preventing loss of follow-45 up; and in developed countries by potentially increasing the participation rate in already well 46 established screening programs by giving women alternatives for screening, which might be 47 more adapted to their busy schedule or budget, encouraging them to get screened. 48 49

2.
Objective 50 Evaluate analytic performance of two transportation and storage devices: Physician collected 51 sample with swab immediately immersed in PreservCyt (dr-WET) versus Self-vaginal 52 sampling with dry swab (S-DRY). 53 54 Women will be invited to perform two samplings for HPV testing. Women will firstly be asked 67 to perform a Self-HPV using a dry swab (S-DRY) and then the physician or nurse will perform 68 a cervical collection immersed in PreservCyt (dr-WET). All specimens will be tested for the 69 same pathogens (HR-HPV) using the same diagnostic test (Xpert HPV). Later, the remaining 70 sample immersed in PreservCyt will be tested for HPV DNA using cobas HPV test. 71

Study procedure: 72
A research nurse will give instructions to the patients and ICF will be obtained. For specimen 73 collection, participants will be instructed to wash their hands before the procedure. Each 74 participant will receive a package containing specimen collection kit. Recommendations will 75 be to hold the swab by the end of the handle, to insert the swab into the vagina, avoiding 76 contact with the external genitalia, until they meet resistance (at least 6 cm). Once they meet 77 resistance, they shall gently turn the swab three to five times. Subsequently the swab shall be 78 inserted in inside a plastic sleeve (S-DRY). Then, the physician or nurse will perform a 79 cervical collection immersed in PreservCyt (dr-WET). In case that the patient is intended to 80 perform a routine cytology during consultation, this cervical collection for the study, will 81 always be performed in second. During colposcopy consultation, the physician or nurse will 82 proceed with the routine consultation. We will look for the histology and cytology results from 83 patients who have done recently a biopsy. 84 At the end, women will complete a self-administered questionnaire on demographics. 85

HPV analysis: 87
Dry swabs (S-DRY samples) will be placed and rinsed into tubes with 3 ml of sterile 88 phosphate-buffered saline (PBS) or NaCl 0.9%, and the tubes will be vortexed for 3×15 sec. 89 Then, 1 ml of each sample will be transferred to the cartridge and will be run on a four-module 90 GeneXpert machine. Tubes containing dr-WET samples (3 ml PreservCyt medium) will be 91 also vortexed for 3×15 sec. Then, 1 ml of each sample will be transferred to the cartridge and 92 will be run on the GeneXpert machine. 93 S-Dry and dr-WET aliquots will be run simultaneously to the GeneXpert machine. The 94 remaining sample immersed in PreservCyt (2ml) will be tested for HPV DNA using cobas 95 HPV test. Once a valid result is obtained, the dry swab specimen will be discarded. 96 Xpert HPV analysis: The Xpert HPV analysis performed consists of real time PCR, using as 97 internal assay control for specimen adequacy, the detection of a human reference gene 98 (HMBS [hydroxymethylbilane synthase]) and an internal Probe Check Control (PCC). The 99 PCC verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye 100

stability. 101
This test includes reagents for the simultaneous detection of 14 hrHPV types (HPV16,18,31,102 33,35,39,45,51,52,56,58,59,66 and 68). 103 The assay utilizes multiple fluorescent channels for the detection of individual types of HPV, 104 groups of HPV, and the human reference gene. Each fluorescent channel has its own cutoff 105 parameters for target detection/validity. If sufficient signal is detected by the human reference 106 gene, the assay results are reported as an overall "positive" if any type of targeted HPV is