Genotype-Specific Distribution of High-Risk Human Papillomavirus (HPV) and Microbial Co-Detections in HPV-Positive Women from Southern Croatia

<b>Background/Objectives</b>: High-risk human papillomavirus (HPV) is the principal etiological agent of cervical cancer, with distinct genotype-specific oncogenic potential. While HPV type 16 is most frequently implicated in carcinogenesis, the role of other genotypes and their interaction with sexually transmitted infections and cervico-vaginal dysbiosis is gaining recognition. This study aimed to assess the genotype-specific distribution of high-risk HPV among HPV-positive women from Southern Croatia and examine associations with age and co-infections with selected microbial pathogens. <b>Methods</b>: We conducted a retrospective cross-sectional study on 1211 HPV-positive women (out of 3098 tested) from Split and Dalmatia County between 2023 and 2024. Cervico-vaginal swabs were tested using molecular and culture-based methods for 14 high-risk HPV genotypes and several pathogens, including <i>Chlamydia trachomatis</i>, <i>Mycoplasma genitalium</i>, <i>Mycoplasma hominis</i>, <i>Ureaplasma urealyticum</i>, <i>Gardnerella vaginalis</i>, and other microorganisms. In the analysis, each detected HPV genotype was also treated as a distinct line-level observation. Genotypes were grouped by phylogenetic and carcinogenic profiles, and statistical analyses-including chi-square tests and multinomial logistic regression-were performed to evaluate associations with age and co-infections. <b>Results</b>: Among high-risk HPV-positive women, the most frequently detected high-risk HPV genotypes were HPV 16 (23.3%), HPV 31 (22.4%), and HPV 51 (13.5%). Notably, HPV 18 was less prevalent (8.1%) and occurred at a similar frequency to HPV 58 and 68. Although younger age was significantly associated with high-risk HPV positivity (<i>p</i> < 0.001), no significant differences in HPV genotype group distribution were observed between age groups; however, <i>C. trachomatis</i> and <i>Streptococcus agalactiae</i> were significantly more prevalent in women aged ≤29 years (<i>p</i> < 0.001 and <i>p</i> = 0.029, respectively). Multinomial regression revealed that <i>C. trachomatis</i> was negatively associated with 16-related and lower-risk genotypes, while <i>G. vaginalis</i> showed a positive association with 16-related types. <b>Conclusions</b>: There is a complex interplay between high-risk HPV genotypes and microbial co-infections, which means the broader cervico-vaginal microbiome has to be considered in HPV risk assessment. The findings highlight the need for extended genotyping and microbial screening to inform regional prevention strategies.