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 diagnostic methods are needed for reliable detection in clinical and veterinary settings. In this study, we evaluated the performance of a commercial indirect immunofluorescence assay test (IFAT) kit originally developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis to determine its ability to detect Encephalitozoon cuniculi and Trachipleistophora hominis in 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 demonstrated comparable mean fluorescence intensity values between E. intestinalis and E. cuniculi, confirming cross-reactive antibody recognition within the Encephalitozoon genus. Serial dilution experiments demonstrated a clear dose-dependent relationship between spore concentration and fluorescence intensity, with linear regression analysis showing a strong positive correlation (R² = 0.9498, P < 0.0001). Sensitivity analysis showed that E. cuniculi could be detected at concentrations as low as 10³ spores/mL. These findings demonstrate that the commercial IFAT kit can reliably detect E. cuniculi despite being designed for other microsporidia species, highlighting its potential utility for broader diagnostic screening of Encephalitozoon infections. Given the zoonotic importance and wide host range of E. cuniculi, this assay may also provide practical value for veterinary and farm animal surveillance, particularly in laboratories with limited access to molecular diagnostic methods.