Concurrent genital-anal human papillomavirus (HPV) infections may impose an increased anal cancer risk in women with HPV-related genital lesions. High viral load may facilitate genital-anal HPV concurrence. Genital and anal HPV is reduced by a bivalent HPV16/18 vaccine, yet the effect on concurrent genital-anal HPV remains unclear. This study analyzed viral load in concurrent genital-anal HPV infections, relative to genital-only and anal-only HPV infections and the impact of vaccination in young women. We included 1074 women, who provided both genital and anal swabs. HPV detection and genotyping was performed using the SPF10-DEIA-LiPA25. HPV copy numbers were measured with type-specific qPCRs and corrected for cellular content to obtain the viral load. Concurrent genital-anal HPV often had significantly higher genital viral load (0.09–371 c/cell) than genital-only HPV (3.17E-04-15.9 c/cell, p < 0.0001 to p < 0.05). Moreover, nearly all concurrent genital-anal HPV types had higher genital copy numbers per PCR reaction (157-416E04 c/rxn) than anal copy numbers (0.90–884E01 c/rxn, p < 0.0001 to p < 0.001). Vaccinated women had significantly less infections with HPV16/18 vaccine-types (2.8% vs 13.7%, p < 0.0001) and HPV31/35/45 cross-protective types (7.4% vs 21.1%, p < 0.0001) than unvaccinated women. In conclusion, particularly high genital viral load is found in concurrent genital-anal HPV infections, which are effectively reduced by vaccination.

Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection systems. The widely used broad-spectrum SPF10-DEIA-LiPA25 (SPF10 method) has reduced sensitivity towards HPV-45 and -59. Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with type-specific (TS) HPV-45 and -59 qPCR assays. The SPF10 method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections, detected by the TS qPCR assays. Viral copy number (VCn) of SPF10 missed HPV-45 and -59 was significantly lower than SPF10 detected HPV-45 and -59 (p<0.0001 for both HPV types). Sanger sequencing showed no phylogenetic distinction between SPF10 missed and detected HPV-59 variants, but variants bearing the A6562G SNP in the SPF10 target region were more likely to be missed (p=0.0392). HPV co-occurrence slightly influenced the detection probability of HPV-45 and -59 with the SPF10 method. Moreover, HPV-59 detection with the SPF10 method was hampered more in non-vaccinated women than vaccinated women, likely due a stronger masking effect by increased HPV co-occurrence in the former group. Consequentially, the SPF10 method led to a strong negative vaccine effectiveness (VE) of -84.6% against HPV-59 while the VE based on TS qPCR was 3.1%. For HPV45, the relative increase in detection in non-vaccinated women compared vaccinated women was more similar, resulting in comparable VE estimates. In conclusion, this study shows that HPV-45 and -59 detection with SPF10 method is dependent on factors including VCn, HPV co-occurrence, and vaccination, thereby showing that knowledge of the limitations of the HPV detection method used is of great importance.

Background Human papillomavirus viral load (HPV VL) is associated with persistence, which increases cervical cancer risk. The bivalent vaccine protects against oncogenic HPV16/18 and cross-protects against several non-vaccine types. We examined the effect of two-dose (2D) and three-dose (3D) vaccination on HPV prevalence and viral load in clearing (CIs) and persistent infections (PIs), 6y and 12y post-vaccination, respectively. Methods Vaginal swabs collected from HPV Among Vaccinated And Non-vaccinated Adolescents (HAVANA, 3D-eligible) and HAVANA-2 (2D-eligble) participants were genotyped for HPV with the SPF10-DEIA-LiPA25. HPV VL was measured with type-specific qPCRs. Results HPV16, 18, 31, 33 and 45 CI and/or PI prevalence and HPV16, 18, and 31 VLs in CIs were significantly reduced in 3D vaccinated women compared to unvaccinated women. Except for HPV11 and HPV59 CIs, no significant VL differences were observed among vaccinated women, ≤6y and &gt;6y post-vaccination. Infection numbers were low in 2D-eligible women, with no HPV16 and 18 in vaccinated women. No VL differences for the remaining types were found. Discussion 3D vaccination reduces HPV prevalence in CIs and PIs and decreases HPV VLs in CIs, 12y post-vaccination for vaccine and several non-vaccine types. 2D-eligible women had low infection numbers, with no HPV16/18 among vaccinated women.