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Journal of Clinical Microbiology, May 2005, p. 2435-2440, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2435-2440.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time PCR for Detection and Identification of Plasmodium spp.

Kathy A. Mangold,1,2 Rebecca U. Manson,2 Evelyn S. C. Koay,3,4 Lindsey Stephens,1 MaryAnn Regner,1 Richard B. Thomson Jr.,1,2 Lance R. Peterson,1,2 and Karen L. Kaul1,2*

Department of Pathology and Laboratory Medicine, Evanston Northwestern Healthcare, Evanston,1 Northwestern University Feinberg School of Medicine, Chicago, Illinois,2 Molecular Diagnosis Centre, National University Hospital, Singapore,3 Department of Pathology, National University of Singapore, Singapore4

Received 16 September 2004/ Returned for modification 9 November 2004/ Accepted 6 January 2005

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.

* Corresponding author. Mailing address: Evanston Northwestern Healthcare, Department of Pathology and Laboratory Medicine, 2650 Ridge Ave., Evanston, IL 60201. Phone: (847) 570-2052. Fax: (847) 733-5012. E-mail: k-kaul{at}northwestern.edu.

Journal of Clinical Microbiology, May 2005, p. 2435-2440, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2435-2440.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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