Development of a multiplex quantitative real-time PCR (qPCR) assay for simultaneous detection of Schistosoma haematobium and high-risk human papillomaviruses

BACKGROUND: Female genital schistosomiasis (FGS) and high-risk human papillomavirus (hr-HPV) infections frequently co-occur in sub-Saharan Africa and may jointly contribute to cervical carcinogenesis. However, no existing molecular platform enables simultaneous detection of Schistosoma haematobium and hr-HPV from a single cervical specimen. METHODS: A multiplex quantitative real-time PCR (qPCR) assay targeting the S. haematobium Dra1 repeat and seven hr-HPV genotypes (16, 18, 31, 33, 45, 52, 58) was developed and validated. Analytical performance was assessed using HPV reference plasmids from the International HPV Reference Centre and biobanked S. haematobium DNA from the Centre for Research in Applied Biology (CeRAB), Ghana. Clinical performance was evaluated using 217 archived cervical swabs from high-risk women enrolled in the CERVIVAL project in rural Ghana. RESULTS: The assay showed a limit of detection of 10 copies/µL for all targets, with no cross-reactivity to non-target HPV genotypes, common uropathogenic bacteria, or Schistosoma mansoni. In reference panels, positive and negative agreement were 98.2% and 100% for S. haematobium and 100% for both sensitivity and specificity across all hr-HPV genotypes. In the 217 high-risk cervical samples, S. haematobium DNA was detected in 56.7% (123/217). HPV58 (19.8%), HPV16 (14.7%), and HPV52 (12.4%) were the most frequent genotypes. Concordance with visual inspection/microscopy for FGS was 93.6% (κ = 0.87), and concordance with a molecular HPV test (ScreenFire HPV) ranged from 90.3% to 99.5% (κ = 0.80-0.95) across HPV channels. The assay identified 11 additional S. haematobium-positive samples that were negative by visual inspection/microscopy. CONCLUSIONS: The multiplex qPCR assay provides a highly sensitive and practical tool for integrated detection of S. haematobium and hr-HPV from a single cervical sample. Its compatibility with standard qPCR platforms and strong agreement with established diagnostic methods support its potential use in FGS and cervical cancer screening programmes in resource-limited, co-endemic settings.