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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 Cryptosporidium parvum is an intracellular parasitic protist that infects both humans and animals (5,22). This parasite belongs to the phylum Apicomplexa. However, recent phylogenetic reconstructions have suggested that the genus Cryptosporidium probably represents an early branch at the base of the phylum (31). Together with C. hominis (a new species recently renamed from type 1 C. parvum) (15), C. parvum is also a significant opportunistic pathogen among immunocompromised individuals (e.g., AIDS patients) (5). Currently, only a single drug (i.e., nitazoxanide [NTZ]) has been approved for the treatment of cryptosporidiosis in the United States and Central and South America (10; see also http://www.romark .com). Therefore, there is a need for the development of new anti-Cryptosporidium drugs.[In vitro drug testing is a critical step in the early stage of drug development. To evaluate drug effects against C. parvum in vitro, parasites must be cultured by infecting human or animal cells. Currently, several techniques are available for the evaluation of drug efficacy against C. parvum in vitro. These techniques include the microscopic method (by counting the number of fluorescently labeled or chemically stained parasites per microscopic field) (8), microtiter enzyme-linked immunosorbent assay (25, 26), chemiluminescence immunoassay (28, 29), and potentially real-time quantitative PCR (qPCR) (6, 13). While all methods may reliably detect the level of parasites in vitro, many have their own limitations. The microscopic method can be labor-intensive and highly subjective. Antibodybased techniques rely on the availability of good antibodies and are limited by the sensitive and limited signal-to-noise ratio (25). More recently, a real-time qPCR-based method has been introduced for assessment of the effects of drugs against C. parvum in vitro by detecting the level of parasite DNA, and this method promised a wide dynamic range of detection (6, 13). However, our recent observations suggest that the DNA level may not always be well correlated with the number of infected parasites because the relationship between the inoculated number of parasites and the normalized cycle thresholds (C T s) of detection by qPCR is not linear. Furthermore, the DNA from dead parasites may also exist in these samples (see below for details). In addition, the reported qPCR method displayed significant plate-to-plate variations (13), indicating that improvements are needed to take full advantage of the real-time PCR system for drug testing.Here we report on an improved method for assessment of drug efficacy against C. parvum infection in vitro, based on the quantitative real-time reverse transcription-PCR (qRT-PCR) technique. By detecting the level ...

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ObjectivePlasma nuclear and mitochondrial DNA (mtDNA) levels are altered in many diseases. However, it is not known whether they are also altered in acute myocardial infarction (AMI). In the present study, we examined plasma nuclear and mtDNA levels in the patients with AMI before and after a percutaneous coronary intervention (PCI) to explore their potential as biomarkers.Methods and resultsPlasma nuclear and mtDNA levels were measured by quantitative PCR in 25 AMI patients, 25 non-myocardial infarction (MI) control participants (with MI risk), and 20 healthy individuals during the study period. The concentrations of nuclear and mtDNA were significantly higher in the AMI group on hospital day 1 than that in the non-MI controls (nuclear: 0.4948±0.0830 vs. 0.2047±0.0222 ng/μl, P<0.05; mitochondrial: 3.754±0.384 vs. 1.851±0.3483 ng/μl, P<0.05) and healthy individuals (nuclear: 0.4948±0.0830 vs. 0.1683±0.0254 ng/μl, P=0.001; mitochondrial: 3.754±0.384 vs. 0.1517±0.0924 ng/μl, P<0.05) and decreased shortly after PCI.ConclusionBoth plasma nuclear and mtDNA levels are elevated in AMI patients, but return to normal levels immediately after PCI, suggesting that they are potentially novel biomarkers for AMI.

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