Density of environmental Acanthamoeba and their responses to superheating disinfection

Exposure to viable Acanthamoeba may cause fatal encephalitis and blinding keratitis in humans. Quantification of environmental Acanthamoeba by a reliable analytical assay is essential to assess the risk of human exposure and efficacy of control measures (e.g., superheating). Two DNA binding dyes (ethidium monoazide (EMA) and propidium monoazide) coupled with real-time quantitative PCR (qPCR) were tested for the ability in selectively quantifying viable Acanthamoeba castellanii. This newly developed qPCR assay was applied to determine the density of environmental Acanthamoeba and disinfection efficacy of superheating. Results showed qPCR with 2.3 μg/mL EMA performed optimal with a great linearity (R (2) = 0.98) and a wide range of detection (5-1.5 × 10(5) cells). EMA-qPCR analyses on water samples collected from cooling towers, eyewash stations, irrigated farmlands, and various wastewater treatment stages further showed viable Acanthamoeba density from nondetectable level to 6.3 × 10(5) cells/L. Superheating A. castellanii at 75-95 °C for 20 min revealed significant reductions in both EMA-qPCR and qPCR detectable Acanthamoeba target sequences with an adverse association between heating temperature and qPCR-determined DNA quantity (r = -0.76 to -0.93, p < 0.0001). Moreover, A. castellanii trophozoites were more sensitive to superheat stress than the cells being encysted for 6 and 13 d (p < 0.05). This is the first study to quantify environmental Acanthamoeba and characterize their responses to superheating by EMA-qPCR. The quantitative data provided in this study facilitate to understand better the relative risk for human exposed to viable Acanthamoeba and the efficacy of superheating against Acanthamoeba.