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Journal of Clinical Microbiology, April 2009, p. 975-980, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.01858-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections{triangledown}

Sandra E. Shokoples,1 Momar Ndao,2 Kinga Kowalewska-Grochowska,3 and Stephanie K. Yanow1*

Provincial Laboratory for Public Health, Edmonton, Alberta, Canada,1 National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada,2 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada3

Received 25 September 2008/ Returned for modification 22 December 2008/ Accepted 15 February 2009

The implementation of real-time PCR for the diagnosis of malaria has been hampered by poor sensitivity for the detection of mixed infections. We have optimized a method that enhances the sensitivity of detection of minor species in mixed infections within a single multiplex reaction. Our assay uses species-specific forward primers in combination with a conserved reverse primer and largely overcomes primer competition for the minor species DNA. With a blind panel of clinical samples, we successfully identified the species in 13/16 mixed infections. This assay was further validated with 91 blood samples and demonstrated a specificity and sensitivity for single infections of 100% compared with nested PCR as the "gold standard." This test has been implemented for routine confirmation of malaria species in Alberta, Canada. In comparison with species identification by microscopy, the real-time PCR test demonstrated greater sensitivity for the identification of species causing low-level and mixed infections and for the discrimination of Plasmodium species other than Plasmodium falciparum. Our experience supports a role for real-time PCR in the identification of malarial species in conjunction with microscopy.

* Corresponding author. Mailing address: Provincial Laboratory for Public Health, WMC Room 2B4.59, 8440 112th Street, Edmonton, Alberta, Canada T6G 2J2. Phone: (780) 407-7558. Fax: (780) 407-3864. E-mail: s.yanow{at}provlab.ab.ca

{triangledown} Published ahead of print on 25 February 2009.

Journal of Clinical Microbiology, April 2009, p. 975-980, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.01858-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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