Purpose: To evaluate the impact of multiple human papillomavirus (HPV) infections on the risk of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in subjects with cervical cytological abnormalities. Methods: A cross-sectional study of 3,842 women attending a colposcopy service was carried out. Genotyping of 18 high-risk, seven low-risk, and two undefined-risk HPVs was carried out by the INNO-LiPA genotyping system. Results: The final colposcopic/pathological diagnoses were as follows: 1,933 (50.3 %) subjects were negative; 1,041 (27.1 %) CIN1; 280 (7.3 %) CIN2; 520 (13.5 %) CIN3; and 68 (1.8 %) invasive cervical cancer. The prevalence of HPV infection was 75.8 % (2,911/3,842), whereas multiple HPVs were detected in 34.5 % of HPV-positive subjects (2,255/3,842). The adjusted risks of CIN3+ in the group with multiple compared to the group with single infection were 2.31 (95 % CI = 1.54–3.47), among HPV16-positive women, and 3.25 (95 % CI = 2.29–4.61, p = 0.21 compared with HPV16-positive subjects), in HPV16-negative subjects. Out of a total of 1,285 subjects with mild lesions, followed up for a median of 16.1 months (interquartile range = 8.9–36.8), the rate of progression to CIN2–3 was 0.6 % (5/541) among subjects negative or with low-risk HPVs, 1.7 % (8/463) among those with single high-risk HPV, and 5 % (14/281, p < 0.001 compared with HPV-negative/low-risk HPV and p = 0.038 compared with single high-risk HPV) among those with multiple high-risk HPVs. Conclusions: Among women with cervical cytological abnormalities, infection by multiple high-risk HPVs increased the risk of CIN3+ in both HPV16-positive and HPV16-negative subjects. These findings suggest a potential synergistic interaction between high-risk HPVs, favoring the progression of CIN lesions.
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