title: Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro.
abstract: We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (C(T)([P18S])) and host cells (C(T)([H18S])), and evaluates the relative expression between parasite and host rRNA levels (i.e., deltaC(T) = C(T)([P18S]) - C(T)([H18S])) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (deltadeltaC(T)) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in approximately 3 h) in dead parasites is faster than that of DNA (99% in approximately 24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 micro/ml and 89.7 to 119.0 microg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, Texas 77843, USA.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Cai
authors.forename: Keith M
authors.initials: KM
authors.lastname: Woods
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Upton
authors.forename: Guan
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000818:Animals; D016785:Cryptosporidium parvum / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D011720:Pyrazoles / Q000494:pharmacology; D016053:RNA, Protozoan / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D011720:Pyrazoles; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; C031280:pyrazole