Development, Validation, and Implementation of an Augmented Multiwell, Multitarget Quantitative PCR for the Analysis...

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established; however, its use as a primary screening option or as a reflex test after atypical cytology results is now gaining wider acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. We developed a new analysis method for the RIATOL quantitative PCR assay, and validated and implemented it in the laboratory of clinical molecular pathology at Algemeen Medisch Laboratorium (AML), under national accreditation and following the International Organization for Standardization guidelines. This study presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.