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We compared the diagnosis of malaria in 297 patients from Thailand by a real-time polymerase chain reaction (PCR) assay using the LightCycler with conventional microscopy using Giemsa-stained thick and thin blood films. The PCR assay can be completed in one hour and has the potential to detect and identify four species of Plasmodium in a single reaction by use of melting temperature curve analysis (however, we did not detect Plasmodium ovale in this study). Blood was collected, stored, and transported on IsoCode STIX, which provide a stable matrix for the archiving and rapid simple extraction of DNA. A genus-specific primer set corresponding to the 18S ribosomal RNA was used to amplify the target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing basepair mismatches, which allowed differentiation of the other Plasmodium species. The PCR results correlated with the microscopic results in 282 (95%) of 297 patient specimens. Most of these were single-species infections caused by P. vivax (150) and P. falciparum (120), along with 5 P. malariae, 2 mixed infections (P. falciparum and P. vivax), and 5 negative specimens. No negative microscopy specimens were positive by PCR (100% specificity for detection of any Plasmodium). The 15 discrepant results could not be resolved, but given the subjective nature of microscopy and the analytical objectivity of the PCR, the PCR results may be correct. The ability of the PCR method to detect mixed infections or to detect P. ovale could not be determined in this study. Within the limitations of initial equipment costs, this real-time PCR assay is a rapid, accurate, and efficient method for the specific diagnosis of malaria. It may have application in clinical laboratories, as well as in epidemiologic studies and antimalarial efficacy trials.
Received January 31, 2005. Accepted for publication June 20, 2005.
Financial support: This study was supported in part by a grant from Mahidol University.
Disclosure: Lynne Sloan and Jon Rosenblatt wish to disclose that they may receive royalties from Roche Applied Science for the development of PCR assays, but not for the malaria assay described in this paper. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.
* Address correspondence to Jon Rosenblatt, Division of Clinical Microbiology, Mayo Clinic, Rochester, MN 55905. E-mail: rosenblatt.jon{at}mayo.edu
Authors’ addresses: Heather Swan, Lynne Sloan, and Jon Rosenblatt, Division of Clinical Microbiology, Mayo Clinic, Rochester, MN 55905, E-mails: swan.heather{at}mayo.edu, sloan.lynne{at}mayo.edu, and rosenblatt.jon{at}mayo.edu. Anthony Muyombwe, Laboratoire de Santé Publique du Quebec, Sainte-Anne-de-Bellevue H9X 1C8, Quebec, Canada. Porntip Chavalitshewinkoon-Petmitr, Srivicha Krudsood, Wattana Leowattana, Polrat Wilairatana, and Sornchai Looareesuwan, Department of Protozoology and Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
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