High-frequency wastewater monitoring of oncogenic human papillomaviruses: a one-year study in Paris

Published: 19 August 2026| Version 1 | DOI: 10.17632/nvb9drxmbs.1
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Description

Background Human papillomaviruses (HPVs) cause nearly 30% of infection-related cancers worldwide. In France, 6,400 cancers annually are attributable to HPV, but surveillance is limited to cervical screening, restricting understanding of population-level HPV genotype circulation. We evaluated whether wastewater-based epidemiology (WBE) could provide a complementary, non-invasive approach for monitoring HPV genotype diversity and temporal trends. Methods Raw wastewater samples were collected twice weekly from a major wastewater treatment plant in Paris between January 2024 and January 2025. Alphapapillomaviruses were quantified using a broad-spectrum quantitative PCR assay. Twenty-eight high-risk and low-risk HPV genotypes were detected using the Allplex™ HPV28 multiplex PCR. Viral signals were normalised using human-associated markers (crAssphage and human mitochondrial DNA). Temporal trends were analysed using statistical modelling accounting for sampling variability and assay detection limits. Findings Alphapapillomaviruses were detected in all samples, with a mean concentration of 7·2.103 genome equivalent per litre. Twenty-seven genotypes were identified, with a median of six genotypes per sample. The most prevalent genotypes were HPV66 (97%), HPV6 (94%), and HPV53 (89%). Oncogenic HPV39 and HPV56, not included in current vaccines, were detected in more than half of samples, whereas vaccine-targeted HPV16 and HPV18 were infrequently detected (19% and 4%, respectively). Temporal analyses indicated stable circulation of HPV6 and HPV66. Interpretation WBE enables population-level monitoring of HPV genotype circulation and could complement clinical surveillance, support evaluation of vaccination programmes, and inform public health decision-making. Funding This work was supported by the Agence Nationale de la Recherche (ANR-24-MIEM-0004) and ANRS-MIE (ANRS00834-R-PR).

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Samples were collected twice weekly from January 24, 2024, to January 29, 2025, using 24-hour composite sampling at the inlet of the Seine Centre WWTP, which serves the Paris metropolitan area (1 million population equivalent). Samples were stored at −20 °C until analysis. Plasmids containing the complete viral genome of 28 HPV genotypes were kindly provided by the International Human Papillomavirus Reference Center, Karolinska Institutet, Sweden. Plasmids were amplified following bacterial transformation (XL1-Blue strain) and purified using the Qiagen Plasmid Midi Kit. Synthetic oligonucleotides encompassing the target sequence were used for genotypes HPV 26, 40, 43, and 70. A 150 mL aliquot of raw wastewater was clarified by centrifugation (3,500 g, 10 min, 4°C). Samples were concentrated using Centricon™ Plus-70 10 kDa units (Merck), yielding approximately 350 µL of samples. The membrane was washed sequentially with PBS, lysis buffer (pH 8), and combined with the concentrate to obtain a final 10 mL lysate as previously described. Nucleic acids were extracted using magnetic silica beads and eluted in 50 µL of elution buffer (Tris-HCl-EDTA, pH 9). PCR inhibitors were removed with the OneStep PCR Inhibitor Removal Kit. Extracts were analysed immediately and after one month of storage at −80°C to evaluate nucleic acid stability and the reproducibility of genotype detection. The SPF10-WW assay was developed by adapting the broad-spectrum SPF10 primers to broaden the detection range of alphapapillomaviruses. Primer sequences were optimized with Geneious Prime using reference genomes curated by the International Human Papillomavirus Reference Center. Plasmids encompassing the complete viral genomes of HPV were employed as positive controls, except HPV 26, 40, 43, and 70, for which synthetic oligonucleotides were used as reference targets. Analytical sensitivity was determined with HPV16 plasmid DNA used as the reference standard. CrAssphage and human mtDNA were quantified using previously validated primer sets, with calibration performed using synthetic oligonucleotide standards encompassing the target sequence. PCR inhibition was evaluated using a titrated internal control of inhibition assay based on an artificial target. All qPCR assays were performed with SensiFAST SYBR® No-ROX (Bioline) in a 20 µl final volume. HPV genotyping was conducted using the Allplex™ HPV28 multiplex PCR assay (Seegene). Five microliters of extracted DNA were processed according to the manufacturer’s instructions. According to the manufacturer, genotypes were considered positive for Ct ≤ 43. HPV types 6 and 66 were quantified in this assay using standard curves generated from serial dilutions of reference plasmids containing the full-length genomes of each virus. The LoD and LoQ for both genotypes were established at 40 copies per reaction. Genotyping was performed on nucleic acids immediately after extraction and after one month of storage at –80°C.

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Epidemiology, Wastewater, Human Papillomavirus

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