TvNTPDase4, a surface ectonucleotidase of Trichomonas vaginalis, is a potential immunological marker for the diagnosis of trichomoniasis in women.

Trichomoniasis is the most common non-viral sexually transmitted infection worldwide but remains underdiagnosed due to limitations of current diagnostic methods. Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme involved in purine salvage in Trichomonas vaginalis and represent a potential diagnostic target. In this study, polyclonal antibodies against recombinant rTvNTPDase4 were produced and an indirect ELISA assay was developed and evaluated for parasite detection in urine samples. The assay detected as few as 10 trophozoites/mL and showed no cross-reactivity with vaginal epithelial cells or representative urogenital microbiota. Clinical validation was performed using 73 urine samples from women previously analyzed by multiplex qPCR. Among female samples, the ELISA showed ≥ 99% sensitivity, 100% specificity, and an area under curve (AUC) of 1.0. Immunofluorescence confirmed surface location of TvNTPDase4 in intact trophozoites, whereas purified anti-TvNTPDase4 IgG showed limited inhibition of enzymatic activity. These findings indicate that TvNTPDase4-based immunoassay may be a promising non-invasive approach for laboratory detection of T. vaginalis in urine samples from women.