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 toward HPV-45 and-59. Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with typespecific (TS) HPV-45 and-59 real-time quantitative PCR (qPCR) assays. The SPF10 method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections that were detected by the TS qPCR assays. The 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 SPF10-detected HPV-59 variants, but variants bearing the A6562G single-nucleotide polymorphism (SNP) in the SPF10 target region were more likely to be missed (P_0.0392). HPV cooccurrence 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 nonvaccinated women than vaccinated women, likely due to a stronger masking effect by increased HPV cooccurrence in the former group. Consequently, 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 HPV-45, the relative increase in detection in nonvaccinated women compared vaccinated women was more similar, resulting in comparable VE estimates. In conclusion, this study shows that HPV-45 and-59 detection with the SPF10 method is dependent on factors including VCn, HPV cooccurrence, and vaccination, thereby showing that knowledge of the limitations of the HPV detection method used is of great importance.