Mixed vaginal infection status in women infected with Trichomonas vaginalis: comparison of microscopy method and metagenomic sequencing analysis

Trichomonas vaginalis (TV) infection is a common non-viral sexually transmitted infection, often combined with mixed vaginal infections. These mixed infections worsen inflammation, disrupt vaginal microbiota, and affect treatment. Currently, TV and its mixed infections are mainly diagnosed by wet mount microscopy, which has low sensitivity and cannot identify complex microbes well. This study compared microscopy with metagenomic sequencing to explore vaginal microbiota changes and improve diagnosis of TV-related mixed infections. We enrolled 30 participants: 20 TV-infected patients (diagnosed by wet mount microscopy) and 10 healthy controls (with Lactobacillus as dominant vaginal microbiota). Then tested by Gram staining, microscopy, and metagenomic sequencing. We analyzed microbial composition and identified different abundant taxa. We also measured clinical indices (Lactobacillus grade, vaginal pH, Nugent score for BV, Donders score for AV) to assess vaginal microecology. Among 20 TV patients, microscopy and clinical criteria found a 65% mixed infection rate (13/20), including TV+AV (5 cases), TV+BV+AV (7 cases), and TV+VVC (1 case). Metagenomic sequencing showed TV patients had higher alpha diversity (Shannon index: p=0.0276) and different beta diversity (ANOSIM, r=0.21, p=0.000167) than controls. At the genus level, TV patients had more anaerobic taxa (Fannyhessea, Atopobium, Peptostreptococcus, FDR<0.05) and less Lactobacillus (FDR<0.05) than controls. All TV patients were CST IV (low Lactobacillus, high mixed bacteria), including 12 cases of CST IV-C and 7 cases of CST IV-B. Microscopy and sequencing had low diagnostic consistency in diagnosing mixed infections, especially for mixed vaginitis. TV infection causes significant vaginal microecological imbalance (less Lactobacillus, more anaerobes, high mixed infection rate). Metagenomic sequencing is better than microscopy at identifying complex microbes and low-abundance pathogens, making it more accurate for diagnosing TV-related mixed infections. These results suggest molecular diagnostic methods should be used as complementary tools for precise analysis improve TV and its mixed infection diagnosis and treatment.