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Antimicrobial Agents and Chemotherapy, November 2005, p. 4437-4442, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4437-4442.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, Texas 77843,¹ Division of Biology, Kansas State University, Manhattan, Kansas 66506²

Received 25 May 2005/ Returned for modification 4 June 2005/ Accepted 2 August 2005

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., C_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 (C_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 3 h) in dead parasites is faster than that of DNA (99% in 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 µg/ml and 89.7 to 119.0 µg/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.

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