Expanded Evaluation of a Commercial Immunofluorescence Kit for Detecting Microsporidia In Vitro
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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 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 demonstrated comparable mean fluorescence intensity values between E. intestinalis and E. cuniculi, consistent with 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 under the in vitro conditions evaluated, despite being designed for other microsporidia species, highlighting its potential utility as a screening tool for Encephalitozoon species. Given the zoonotic importance and wide host range of E. cuniculi, this assay may provide a practical screening approach for veterinary and farm animal surveillance following further validation in biological specimens, particularly in laboratories with limited access to molecular diagnostic methods.
