Expanded Evaluation of a Commercial Immunofluorescence Kit for Detecting Microsporidia In Vitro

Microsporidia are important opportunistic intracellular parasites that infect both humans and animals, and rapid detection methods are important for their identification in clinical and veterinary settings. In this study, we evaluated the performance of a commercial indirect immunofluorescence assay test (IFAT) kit originally developed for detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis to determine its ability to detect Encephalitozoon cuniculi and Trachipleistophora hominisin vitro. Rabbit kidney epithelial (RK-13) cells were infected with E. intestinalis, E. cuniculi, or T. hominis and analysed using fluorescence microscopy and quantitative fluorescence intensity analysis. Strong fluorescence signals were observed in both E. intestinalis and E. cuniculi infected cells, whereas T. hominis showed no detectable fluorescence. Quantitative fluorescence intensity analysis revealed similar mean fluorescence intensities for E. intestinalis and E. cuniculi, consistent with potential cross-reactive antibody recognition within the Encephalitozoon genus. Serial dilution experiments demonstrated a concentration-dependent relationship between spore concentration and fluorescence intensity, with linear regression analysis showing a strong positive correlation (R² = 0.9498, P < 0.0001). The lowest concentration at which fluorescence was observed was 10³ spores/mL, with positive signals detected in 30% of examined fields. These findings support the detection of E. cuniculi using the commercial IFAT kit under the in vitro conditions tested, despite being designed for other microsporidia species, suggesting its potential applicability to other Encephalitozoon species. Given the zoonotic importance and wide host range of E. cuniculi, this assay may have potential applications in veterinary surveillance, following further validation in biological specimens, particularly in laboratories with limited access to molecular diagnostic methods.