Investigating the Role Played by Trichomonas vaginalis in Promoting Chlamydial Spread to the Upper Genital Tract

<strong>Introduction:</strong> <em>Chlamydia trachomatis</em> is the most common bacterial STI in the U.S. and can lead to serious reproductive health issues if it ascends to the upper genital tract. While co-infections with pathogens like <em>Neisseria gonorrhoeae</em> have been linked to more severe disease, a role for <em>Trichomonas vaginalis</em> in <em>C. trachomatis</em> ascension remains unclear. <em>T. vaginalis</em>, a protozoan pathogen, can damage genital tissues and increase susceptibility to other infections. Recent research suggests that <em>T. vaginalis</em> virulence is enhanced by endosymbionts, such as <em>Mycoplasma hominis</em> and <em>Candidatus </em>Mycoplasma girerdii. <strong>Methodology:</strong> The TRAC 2 study utilized a longitudinal cohort and cross-sectional evaluation to investigate host immune responses and risk factors associated with <em>C. trachomatis</em> infection. Between November 2019 and June 2023, 120 women aged 15&ndash;40 were recruited from an Allegheny County public health clinic. Eligibility was based on clinical presentation or known exposure to sexually transmitted infections. Cervical and vaginal samples were collected and preserved in DNA/RNA Shield, with nucleic acids extracted using Zymo DNA/RNA Miniprep Kits. Quantitative PCR targeting the &beta;-tubulin gene of <em>T. vaginalis</em> was conducted using SYBR Green chemistry and validated with a standard curve using template obtained from a previously characterized clinical specimen. Triplicate reactions, no-template controls, and inter-plate standards were used to ensure reliability and to control for batch effect. Plate maps were designed to distribute clinical profiles evenly and minimize technical variation. <strong>Results: </strong>The TRAC 2 cohort, consisting of 120 women, had a mean age of 24.59 years (SD = 5.15), with 65.0% of participants identifying as Black and 93.3% not Hispanic or Latino. A significant portion of the cohort was uninsured (17.5%) or covered by Medicaid (38.3%). Behavioral health data revealed high rates of alcohol consumption (80.8%) and drug use (75.8%). Gynecologic history showed that 48.2% had ever been pregnant, with 56.8% having a history of chlamydia. Pathogen prevalence revealed cervical chlamydial infection in 78.4%, and trichomonas in 11.2% of participants. qPCR analysis indicated significantly higher signal intensities in clinically confirmed T. <em>vaginalis</em> (TV+) cases, with average values of 1.78E+11, compared to 3.35E+10 in TV&minus; cases. Symptoms such as pain or burning and frequent urination were more common among TV+ individuals, while abnormal discharge was more frequently reported in TV&minus; participants. Overall, TV+ individuals reported fewer symptoms when compared to TV&minus; individuals, indicating that the TV+ group had a lower symptom burden. Additionally, individuals with motile Trichomonas exhibited a higher average burden but fewer symptoms than those without motile Trichomonas. <strong>Discussion: </strong>Our data provide preliminary evidence that <em>T. vaginalis</em> abundance may be inversely associated with <em>C. trachomatis</em> ascension, echoing previous findings implicating <em>T. vaginalis</em> endosymbionts in modulating genital tract inflammation. While causality cannot be established from this cross-sectional analysis, the results support a potential protective or competitive interaction between <em>T. vaginalis</em> and <em>C. trachomatis</em> that may influence the likelihood of upper genital tract involvement. These findings raise new questions about pathogen-pathogen interactions and highlight the need for mechanistic studies exploring how <em>T. vaginalis</em> and its symbionts may alter epithelial immunity and <em>C. trachomatis</em> pathogenesis.