Detection of Genital Pathogens and Co-Infections by Multiplex RT-qPCR: Associations with HIV Positivity and Demographic Factors

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Abstract
Background/Objectives: This study aimed to investigate the genital pathogen profile and co-infection dynamics using multiplex RT-qPCR, specifically evaluating the independent associations with age, sex, and HIV status. Methods: Data from 1217 patients who underwent a sexually transmitted infection (STI) panel study at the Microbiology Laboratory of Marmara University Pendik Training and Research Hospital between January 2024 and December 2025 were retrospectively reviewed. Pathogen detection was performed using a commercial kit (Bioeksen, Istanbul, Türkiye) with the multiplex RT-qPCR method. The independent effects of HIV positivity, age, and sex on pathogen frequency and co-infection were analyzed using logistic regression models; results were evaluated using adjusted odds ratio (aOR) and 95% confidence interval (CI). Results: Any-pathogen positivity was detected in 57.8% of patients, with Ureaplasma spp. (36.9%) and Gardnerella vaginalis (32.5%) being the most prevalent. While overall pathogen positivity did not differ significantly by HIV status (p = 0.158), HIV-positive patients exhibited distinct microbiological architectures. Higher positivity rates were observed in women in both groups, and a strong correlation was found between sex and the presence of infection (p < 0.001). Multivariate analysis revealed that HIV positivity was independently associated with an over fivefold increase in HSV-2 detection (aOR = 5.09, 95% CI: 1.47–17.65; p = 0.010). Furthermore, HIV-positive individuals were significantly enriched for complex polymicrobial patterns involving three or more pathogens (p = 0.008). Conversely, male sex was independently associated with a substantially lower risk of co-infection (aOR = 0.17, 95% CI: 0.12–0.22; p < 0.001), and increasing age showed an inverse relationship with co-infection frequency (aOR = 0.98 per year, 95% CI: 0.97–0.99; p = 0.001). Conclusions: This study adds to current epidemiological evidence by showing that genital pathogen distribution is shaped by age- and sex-related heterogeneity, with flora-associated co-infections predominating in women and more classical STI-related agents occurring more often in men. Our findings suggest that multiplex RT-qPCR provides value not only for broad pathogen detection but also for identifying demographic- and HIV-associated co-infection patterns that may support stratified screening and targeted clinical management.

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