Combination of cell culture and quantitative PCR for screening of drugs against Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic pathogen causing self-limiting diarrhea in immunocompetent patients. An assay combining cell culture and real time quantitative PCR (qPCR) is reported here to verify drug efficacy against C. parvum in vitro. The monolayers of Human ileocecal adenocarcinoma cells (HCT-8) were infected by sporozoites excysted directly on the cells and were incubated with monensin, halofuginone bromide and hexadecylphosphocholine until 45h post infection (p.i.). The genomic DNA was extracted at 3, 27 and 45h p.i. and subjected to qPCR targeting the 70kDa heat shock protein gene to quantify the development of C. parvum. The reliability of the method was validated by testing of monensin and halofuginone bromide, which are well known to be effective in vitro. With the dose dependency monensin and halofuginone showed a maximum inhibition of 98.15% and 98.05% at 0.144 and 25microM, respectively, compared with non-treated controls at the endpoint incubation, confirming previous reports. The reduction of the parasite DNA reproduction over 27h p.i. compared with 3h p.i. was found to be as 97-99% in 0.144microM monensin and 99% in 25microM halofuginone treated cells. The new antileishmanial compound hexadecylphosphocholine (24.5microM, Miltefosine) showed 78-98% inhibition at 45h p.i., however, the reproduction of parasite DNA was reduced to 96-98% over 27h p.i. The method has the potential to easily and reliably assess anticryptosporidial compounds in adequately equipped routine laboratories.