Imaging characteristics and comparative evaluation of dual-fluorescence assay in various types of vaginitis: a cross-sectional study

Rapid and accurate differentiation of common vaginitis remains challenging in routine clinical practice. Dual-fluorescence assay, which simultaneously visualizes microorganisms and host cells under blue light excitation, may serve as an adjunctive tool to conventional microscopy. This study explored the imaging characteristics of dual-fluorescence staining in cultured vaginal epithelial cells and standard reference microbial strains, and further evaluated its diagnostic performance in a clinical population of 758 women undergoing vaginal secretion testing. (1) Under 450-500 nm blue light excitation: Red fluorophores specifically labeled microorganisms (including pathogenic fungi, bacteria, and Trichomonas vaginalis), while green fluorophores counterstained host cells (e.g., epithelial cells and leukocytes). The distinct color contrast between microorganisms and host cells, combined with microbial morphological features, enabled rapid localization and identification of pathogens. (2) Dual-fluorescence assay demonstrated strong concordance with conventional microscopy: For bacterial vaginosis (BV), the sensitivity was 0.886 (95% CI: 0.796-0.955), specificity was 0.958 (95% CI: 0.931-0.979), and Kappa was 0.821 (95% CI: 0.756-0.901); for vulvovaginal candidiasis (VVC), sensitivity was 0.953 (95% CI: 0.840-0.988), specificity was 0.978 (95% CI: 0.953-0.990), and Kappa was 0.908 (95% CI: 0.847-0.963); for aerobic vaginitis (AV), sensitivity was 0.800 (95% CI: 0.535-0.848), specificity was 0.959 (95% CI: 0.924-0.995), and Kappa was 0.697 (95% CI: 0.616-0.863); and for trichomoniasis (TV), sensitivity was 0.833 (95% CI: 0.365-0.991), specificity was 0.995 (95% CI: 0.979-0.999), and Kappa was 0.766 (95% CI: 0.331-1.000). The dual-fluorescence assay technique enables simultaneous detection of four common types of vaginitis (including mixed vaginitis) while integrating the diagnostic advantages of both wet mount and Gram staining methods, thus demonstrating high clinical utility.