A-262 Optimizing Laboratory Diagnostics for Papillomavirus Prevention: Which Molecular Biology Methods for Screening?

Abstract Background Despite advancements in precision screening technologies, HPV testing continues to be a rapidly growing area of research worldwide. The present study correlates two molecular methods for HPV DNA testing and two analytical instruments with the aim of identifying the most sensitive and specific method, respecting the Italian Carcinoma Screening Group (GISci) guidelines for the reporting of molecular screening test and possible extended genotyping of positive patients. Methods Specimens were collected from 48 female patients, aged 26 to 66 years (mean age 45), at an outpatient gynecology clinic and analyzed at the Laboratory of Clinical Analysis, Humanitas Mater Domini, Castellanza, Varese, Italy. Specimens were placed in ThinPrep™ Pap-Test PreservCyt™ containers (Hologic, Italy) and analyzed using two instruments: INNO-Lipa 48 (Fujirebio®) [Method 1] and Hybridspot 12 PCR AUTO (Eurospital, Italy) [Method 2], with confirmation by CFX96TM Dx System (Bio-Rad, Italy) [Method 3]. Three diagnostic kits were used for amplification: INNO-Lipa HPV Genotyping Extra II Amp kit (Fujirebio®), HPV Direct Flow Chip kit (Vitro Master Diagnostics®), and NeoPlex HPV 29 (Genematrix Inc.®). Method 1 detects 32 HPV genotypes (18 HR, 14 LR), and Method 2 detects 35 genotypes (18 HR, 17 LR). Results Method 1 analysis showed 5 negative (10%) and 43 positive (90%) samples: 28 (65%) HR-genotypes only, 9 (21%) LR-genotypes only, and 6 (14%) both. Method 2 showed 11 negative (23%) and 37 positive (77%) samples: 15 (43%) HR-genotypes, 8 (19%) LR-genotypes, and 14 (38%) both. Of 48 samples, 23 were discordant. In Group A, 10 samples (43%) were positive with one method but negative with the other: Method 1 identified 8 positives and 2 negatives, while Method 2 identified 2 positives (HR-genotypes 56 and 59) and 8 negatives. Group B had 13 discordant samples, with Method 1 identifying more HR-genotypes and Method 2 identifying more LR-genotypes. Method 3 confirmed 8 positives and 2 negatives in Group A, with 6 Method 1 positives and 2 Method 2 positives as true positives. In Group B, Method 3 identified HR-genotypes in all 13 samples, with 3 and 5 discordant from Method 1 and Method 2, respectively. It identified LR-genotypes in 9 samples, with 6 discordant from Method 1 and 5 from Method 2. Among the 25 concordant samples, 11 analyzed with Method 3 showed 100% agreement across all methods. Conclusion Molecular detection of HPV DNA, RNA, or proteins has limitations but remains effective when properly evaluated for clinical sensitivity and specificity. A user-friendly method that reduces variability during sample preparation, amplification, and genotyping is crucial for standardization, minimizing human error and improving accuracy. The Vitro kit offers these benefits, reducing reporting time and improving patient care. Based on sample data, despite Method 2 being nearly fully automated and Method 1 requiring more operator input, Method 1 is superior for screening and extended genotyping of HR-genotypes, while Method 2 is better for LR-genotypes, offering faster response times and higher repeatability without compromising result quality.