Point-of-Care Tests to Amplify and Detect High-Risk HPV DNA and mRNA
Kundrod, Kathryn Ann
Doctor of Philosophy
Cervical cancer is a major global health challenge, with 570,000 new diagnoses and 311,000 deaths annually. The burden of cervical cancer remains highest in resource-limited settings, where screening, diagnosis, and treatment are challenging to implement and sustain. The preferred method of cervical cancer screening is testing for high-risk human papillomavirus (HPV), the etiologic agent that causes cervical cancer. HPV DNA testing is the most sensitive method of cervical cancer screening, and HPV mRNA testing has the potential to maximize clinical sensitivity and specificity by detecting infections that are progressing toward cancer. Currently available methods of HPV DNA and mRNA testing remain too costly or the instrumentation remains too complex for effective use at the point of care in resource-limited settings. This work presents the development of two tests to detect high-risk HPV DNA and mRNA with lower cost and instrument complexity relative to existing tests. The developed tests detect two HPV genotypes—HPV 16 and 18—that cause approximately 70% of invasive cervical cancer cases globally. First, this work describes an HPV 16 and 18 DNA test that is integrated from sample to answer, combining sample preparation, isothermal amplification, and lateral flow detection. The testing workflow involves six user steps and produces a visual result within 45 minutes. In a set of 30 cellular samples collected with cervicovaginal swabs, the developed test reliably detected samples with >1,000 copies of HPV 16 or 18 DNA per reaction, which is a clinically relevant limit of detection. The second part of this work describes a low-cost, low-infrastructure method of HPV mRNA detection. The test relies on one-step reverse transcription and isothermal amplification with real-time fluorescence readout, allowing for semi-quantitative detection. A clinically relevant analytical sensitivity of 1,000 HPV 16 or 18 E7 gene transcripts per reaction in extracted cellular RNA was demonstrated. Together, the developed tests are a critical step toward point-of-care cervical cancer screening through HPV DNA and mRNA testing. With future work to incorporate additional genotypes and optimize sample preparation strategies, the developed tests have the potential to increase access to high-quality cervical cancer screening globally.
Point-of-care tests; HPV; cervical cancer; global health; resource-limited settings