Improvement of in vitro evaluation of chemical disinfectants for efficacy on Cryptosporidium parvum oocysts.

Cryptosporidium parvum has been suggested as a suitable target for in vitro efficacy testing of disinfectants. To improve validity of a method based on exposure of HCT-8 monolayers to C. parvum oocysts we here critically evaluate and we propose certain procedural steps needed for the validation of disinfectants. Within a range of 0.02% to 0.4%, sodium taurocholate at 0.2% stimulated infection most efficiently while preserving host cell integrity. The course of invasion was monitored for periods of 30-240min post infection (p.i.). FACS analysis revealed that the proportion of sporozoites liberated from oocysts in the presence of 0.2% sodium taurocholate increased within 120min of incubation but remained constant thereafter. Maximum invasion of cells measured by qPCR was reached 180min p.i. and therefore set as invasion endpoint. As monolayers harvested 24h or 48h p.i. did not differ in the quantity of parasite hsp70 gene copies, DNA extraction can be performed as early as 24h p.i. Incubation of oocysts with 20% HO for 2h resulted in inactivation of more than 99.5% both at room temperature and 10°C and appeared thus suitable as positive chemical treatment control. Four washing procedures considered to remove potentially toxic residual disinfectant from oocyst suspensions were tested. An application of a combination of DMSO (Dimethylsulfoxid), Tween20 and WSH (water of standardized hardness) appeared most efficient without deleterious effect of disinfectant residuals on the cell monolayer viability when oocysts accordingly washed were applied. In conclusion, for standardized in vitro evaluation of chemical disinfectants in C. parvum infected HTC-8 monolayers. (i) excystation medium should contain 0.2 % sodium taurocholate. (ii) excystation medium should be replaced by growth medium after 180 min. (iii) monolayers should be harvested 24 h p.i. for DNA preparation. (iv) ocysts exposed to 20 % HO should be included as positive controls. (v) disinfected oocysts should be washed with DMSO/Tween20/WSH before they are transferred to monolayers.