Modified wet mount staining improves the microscopic diagnosis of Trichomonas vaginalis in vaginal discharge

Conventional microscopic diagnosis of Trichomonas spp. in vaginal discharge specimens relies entirely on organism motility. Herein, we established a novel modified methyl green-acid fuchsin staining method to differentiate Trichomonas vaginalis from leukocytes and epithelial cell nuclei based on staining characteristics, rather than motility. To evaluate the performance of this novel method, 2133 vaginal discharge specimens were subjected to clinical testing using safranin staining microscopy, Trichomonas culture, polymerase chain reaction (PCR), and the proposed method, with an expanded gold standard as the reference. Additionally, the impact of specimen refrigeration (2-8 ℃) on the detection rate of T. vaginalis was also investigated. With the proposed method, motile Trichomonas remained unstained, whereas non-motile Trichomonas appeared light-purple. Leukocytes were stained blue-green, the epithelial cell cytoplasm was light purple, and the epithelial nuclei were blue-green. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of this method were 77.78% (95% confidence interval [CI]: 65.06-86.80), 100.00% (95% [CI]: 99.82-100.00), 100.00% (95% [CI]: 91.62-100.00), and 99.43% (95% [CI]: 99.00-99.67), respectively. For fresh specimens, the proposed method exhibited a statistically significant difference compared with safranin staining microscopy (P < 0.001) and was not affected by 24 h of specimen refrigeration. In contrast, 18 of the 25 initially Trichomonas-positive specimens became undetectable by safranin staining microscopy after refrigeration. The distinct staining characteristics and size differences among cellular components enable the novel wet-mount staining method to morphologically differentiate T. vaginalis, leukocytes, and denuded epithelial nuclei.