Chemical disinfection of Encephalitozoon cuniculi: toward evidence-based infection control guidelines

Background Encephalitozoon cuniculi is an opportunistic pathogen with significant zoonotic potential, particularly for immunocompromised individuals. However, evidence-based disinfection protocols against its environmentally resistant spores are lacking, leading to potential reliance on suboptimal agents. Objective This study aimed to establish a rigorous, data-driven hierarchy for the efficacy of common chemical disinfectants against E. cuniculi spores by integrating assessments of structural integrity, cellular infectivity, and in vivo dissemination. Methods We employed a multi-modal approach. Spores were treated with rapid-acting (75% ethanol, 1% hydrogen peroxide, chlorine-based agent) and long-acting (nano-silver, quaternary ammonium compounds—QACs) disinfectants. Sporicidal effects were evaluated via flow cytometry using ethidium bromide (EB) staining. Functional infectivity was quantified in Vero cells using a species-specific TaqMan qPCR assay targeting intracellular spore DNA. A murine model was used to assess the capacity of treated spores to establish systemic infection, quantified by spore DNA load in blood and kidney tissue. Results Flow cytometry revealed that 75% ethanol caused significant membrane damage (~51% EB-positive spores in 20 min) but failed to consistently abolish infectivity in cell culture or mice. Chlorine-based agents and hydrogen peroxide demonstrated potent, time-dependent inactivation, achieving >96 and 100% infectivity reduction in cells and mice, respectively, within 60 min. Notably, chlorine agents rendered spores non-infectious without inducing proportional EB uptake, indicating a mechanism distinct from gross membrane disruption. The long-acting disinfectant QACs necessitated an extended contact time (up to 5 h) to achieve complete prevention of in vivo infection. This prolonged exposure induced substantial spore aggregation, thereby impeding accurate flow cytometric analysis. Conclusion The chlorine-based disinfectant and hydrogen peroxide (rapid-acting), as well as the quaternary ammonium compound (long-acting), all effectively eliminated the infectivity of E. cuniculi spores. Crucially, we demonstrate a critical dissociation between spore structural damage and viability, highlighting the necessity of functional infectivity assays over structural metrics alone. This study provides a foundational framework for evidence-based infection control guidelines in clinical, veterinary, and public health settings.