Performance of DiaRD-HPV Rt-PCR Kit for Detection of High-Risk Human Papilloma Virus Genotypes: A Preliminary Study

Abstract

Objectives: Rapid detection and identification of high-risk human papillomavirus (hrHPV) genotypes in cervical specimens has a crucial importance for management of patients. In this study, we evaluated the performance of the novel DiaRD-HPV Rt-PCR kit developed for screening HPV in cervical samples and identifying hrHPV types 16 and 18. Materials and Methods: The performance of the novel DiaRD-HPV Rt-PCR kit was tested on a total of 243 clinical samples, including 65 HPV positive (genotype 16, n=23; genotype 18, n=2; other genotype, n=40) and 178 HPV negative samples previously tested with the QIAscreen HPV PCR test kit. DNA was isolated from cervical specimens in PreservCyt transport medium using the Diarex Viral DNA/RNA extraction kit. Multiplex PCR was performed following the DiaRD-HPV Rt-PCR kit protocol. Results: The results of the novel kit were compared to those of the QIAscreen HPV PCR test kit using a comparator test. Of the 65 samples that were positive with the comparator kit, 62 (95.4%) were also positive with the novel kit. All 178 HPV DNA negative samples were also negative with the novel kit. The sensitivity, specificity, and accuracy of the novel kit were 95.4%, ≥99.9%, and 98.8%, respectively. According to the cycle threshold (Ct) values determined in the reproducibility studies, the intra-assay and inter-assay coefficients of variations (CVs) were less than 1%. Conclusion: The high concordance between the results of the novel kit and comparator kit supports the use of the DiaRD HPV Rt-PCR kit in clinical samples.

Keywords

• Human papillomavirus
• Rt-PCR
• DiaRD HPV kit

References

1. World Health Organization (WHO). Human Papillomavirus (HPV). Last accessed date: 2025 Fab 20. Available from: https://www.who.int/teams/health-product-policy-and-standards/standards-and-specifications/norms-and-standards/vaccine-standardization/human-papillomavirus
2. National Cancer Institute. HPV and Cancer. Last accessed date: 2025 Fab 20. Available from: https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer#what-is-hpv-human-papillomavirus
3. Sua P, Maa I, Yua L, Tanga S, Sun P. Clinical significance of extended high-risk human papillomavirus genotyping and viral load in cervical cancer and precancerous lesions. Gynecol Obstet Clin Med. 2023;3:22–29.
4. Perkins RB, Wentzensen N, Guido RS, Schiffman M. Cervical cancer screening: A review. JAMA. 2023;330(6):547-58.
5. Bruni L, Albero G, Serrano B, Mena M, Collado JJ, Gómez D, et al. ICO/IARC Information Centre on HPV and Cancer (HPV Information Centre). Human Papillomavirus and Related Diseases in the World. Summary Report 10 March 2023. [Accessed: 21 February 2025]
6. Centers for Disease Control and Prevention. Basic Information about HPV and Cancer. Last accessed date: 2025 Fab 20. Available from: https://www.cdc.gov/cancer/hpv/basic-information.html
7. Arbyn M, Simon M, Peeters E, Xu L, Meijer CJLM, Berkhof J, et al. 2020 list of human papillomavirus assays suitable for primary cervical cancer screening. Clin Microbiol Infect. 2021;27(8):1083-95.
8. Williams J, Kostiuk M, Biron VL. Molecular detection methods in HPV-related cancers. Front Oncol. 2022;12:864820.

9. Ouyang Y, Abuduxikuer G, Abulimiti T, Abudurexiti G, Zhuo Q, Li W, et al. Clinical performance of the SureX PCR HPV test versus hybrid capture assays (DH2/careHPV) in primary cervical cancer screening. Tumour Virus Research. 2025;200319, ISSN 2666-6790.
10. Feldstein O, Gali-Zamir H, Schejter E, Feinberg T, Yehuda-Shnaidman E, Bornstein J, Levy T. High-risk HPV testing vs liquid-based cytology for cervical cancer screening among 25- to 30-year-old women: A historical cohort study. Acta Obstet Gynecol Scand. 2023;102(2):226-33.
11. Maver PJ, Poljak M. Primary HPV-based cervical cancer screening in Europe: Implementation status, challenges, and future plans. Clin Microbiol Infect. 2019;26(5):579-83.
12. Pereira AR, Redzic N, Van Vooren S, Pelak K, Broekmans A, Desloovere G, et al. Development, validation, and implementation of an augmented multiwell, multitarget quantitative PCR for the analysis of human papillomavirus genotyping through software automation, data science, and artificial intelligence. J Mol Diagn. 2024;26(9):781-91.
13. Santos FLSG, Invenção MCV, Araújo ED, Barros GS, Batista MVA. Comparative analysis of different PCR-based strategies for HPV detection and genotyping from cervical samples. J Med Virol. 2021;93(11):6347-54
14. Cheung LC, Egemen D, Chen X, Katki HA, Demarco M, Wiser AL, et al. ASCCP risk-based management consensus guidelines: methods for risk estimation, recommended management, and validation. J Low Genit Tract Dis. 2020;24(2):90-101.
15. Yan C, Ma J, Zheng X, Shu J, Wang X, Chen X, et al. Diagnostic accuracy of human papillomavirus testing on self-collected samples among women referred for colposcopy. Sci Rep. 2025;15(1):2513.
16. Lindquist S, Kjær SK, Frederiksen K, Ørnskov D, Munk C, Waldstrøm M. Comparative analysis of HPV testing versus cytology in Danish cervical cancer screening: Insights from a large-scale implementation study. Gynecol Oncol. 2024;191:45-55.
17. QIAscreen® HPV PCR Test Instructions for Use (Handbook). [Last accessed February 20, 2025]. Available from: https://self-screen.nl/wp-content/uploads/2024/06/HB-2579-004_1132289_R3_QIAScreen_PCR_CE_0623_EMEA.pdf
18. Taskin MH, Nursal AF, Oruc MA, Kariptas E. Genotype distribution and prevalence of high-risk human papillomavirus infection among women in Samsun province of Turkey. Asian Pac J Cancer Prev. 2022;23(7):2477-82.
19. Alacam S, Bakir A. Human papillomavirus prevalence and genotype distribution in cervical swab samples in Istanbul, Turkey. J Infect Dev Ctries. 2021;15(8):1190-1196.
20. Beyazit F, Sılan F, Gencer M, Aydin B, Paksoy B, Unsal MA, Ozdemir O. The prevalence of human papillomavirus (HPV) genotypes detected by PCR in women with normal and abnormal cervico-vaginal cytology. Ginekol Pol. 2018;89(2):62-67.
21. Çalişkan E, Coşkun SK, Öztürk CE, Cangür Ş, Önail B. Analysis of HPV genotypes and liquid-based cervical cytology: Results from a tertiary academic center in northwestern Turkey. Jpn J Infect Dis. 2021;74(1):69-72.
22. Tekkesin N, Goktas S, Alkis V, Tekkesin E, Goktas P. Male Human papillomavirus infection and genotyping in Turkey. Asian Pac J Cancer Prev. 2023;24(12):4187-93.
23. Hesselink AT, Berkhof J, van der Salm ML, van Splunter AP, Geelen TH, van Kemenade FJ, et al. Clinical validation of the HPV-risk assay, a novel real-time PCR assay for detection of high-risk human papillomavirus DNA by targeting the E7 region. J Clin Microbiol. 2014;52(3):890-896.
24. Keung MHT, Mehta N, Lynn E, Pineda E, Fetene D, Lee A, et al. Clinical validation of the Roche cobas HPV test on the Roche cobas 6800 system for the purpose of cervical screening and qualification as a second-generation comparator test. J Virol Methods. 2025;335:115145.
25. Martinelli M, Latsuzbaia A, Bonde J, Pedersen H, Iacobone AD, Bottari F, et al. Extended Valhudes Study Group. Performance of BD Onclarity HPV assay on FLOQSwabs vaginal self-samples. Microbiol Spectr. 2024;12(3):e0287223.
26. Latsuzbaia A, Vanden Broeck D, Van Keer S, Weyers S, Tjalma WAA, Doyen J, et al. Clinical performance of the RealTime high risk HPV assay on self-collected vaginal samples within the VALHUDES framework. Microbiol Spectr. 2022:10(5):e0163122.
27. Moyo S, Ramogola-Masire D, Moraka NO, Tawe L, Noubary F, Motsumi K, et al. Comparison of the AmpFire® multiplex HPV assay to the Xpert® HPV assay for detection of human papillomavirus and cervical disease in women with human immunodeficiency virus: A pragmatic performance evaluation. Infect Agent Cancer. 2023;18(1):29.