Optimization and analytical validation of the Allplex HPV28 genotyping assay for use in first-void urine samples

Abstract

Despite first-void urine (FVU) being increasingly recognized as a credible specimen for human papillomavirus (HPV) detection, there is a lack of well-validated testing methods providing full quantitative genotyping required for vaccine impact monitoring from FVU samples. The Allplex HPV28 assay, capable of individually detecting 28 HPV genotypes, presents a promising method. We aimed to evaluate its genotype-specific performance on FVU samples, following optimization of FVU preanalytics. We selected 701 FVU samples collected using a Colli-Pee device (20 mL, with UCM), enriched for HPV-positivity ( n = 630) based on previous testing with GP5+/6+-PCR-based reverse line blot (GP5+/6+ RLB) and E7-MPG after Amicon filtration (AF). We first evaluated the comparability and agreement of Allplex HPV28 genotype-specific positivity according to different preanalytics. Subsequently, we conducted the genotype-specific comparison of Allplex HPV28 with GP5+/6+ RLB AF and E7-MPG AF. No significant differences in HPV positivity by Allplex HPV28 testing were observed when comparing pre-centrifuged versus non-centrifuged DNA extraction, nor when comparing manual versus automated DNA extraction. Good genotype-specific agreement was observed between Allplex HPV28 and GP5+/6+ RLB AF, with Allplex HPV28 being slightly more sensitive for all 28 HPV genotypes (average Allplex HPV28:GP5+/6+ RLB AF ratio 1.729). Compared to E7-MPG AF, Allplex HPV28 exhibited lower sensitivity for all 21 overlapping HPV genotypes (average Allplex HPV28:E7-MPG AF ratio 0.588). The findings of this study, combined with practical considerations for real-world implementation, support the use of Allplex HPV28 testing after automated or manual DNA extraction without the requirement for pre-centrifugation, for HPV studies based on FVU samples, most notably those for vaccine impact monitoring on HPV prevalence. IMPORTANCE This study provides the first analytical validation of the Allplex HPV28 genotyping assay for use in first-void urine samples, offering a reliable, non-invasive, and practical alternative to cervical samples for human papillomavirus (HPV) detection. It demonstrates a validated approach that supports the assay’s potential application in real-world settings, including low- and middle-income countries, where non-invasive and widely acceptable sampling methods are crucial for maximizing population coverage and representativity. Given the urgent need for accurate and practical tools to monitor HPV vaccination impact, these findings offer a timely and impactful contribution to the field.