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Journal of Clinical Microbiology, March 2004, p. 1214-1219, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1214-1219.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time PCR Assay for Detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for Routine Clinical Diagnosis

F. Perandin,1 N. Manca,1* A. Calderaro,2 G. Piccolo,2 L. Galati,2 L. Ricci,3 M. C. Medici,2 M. C. Arcangeletti,2 G. Snounou,4 G. Dettori,2 and C. Chezzi2

Department of Laboratory Diagnosis, Section of Microbiology, University of Brescia, Spedali Civili, 25123 Brescia,1 Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, 43100 Parma,2 Arcispedale S. Maria Nuova, Centrale Operativa, 42100 Reggio Emilia, Italy,3 Unité de Parasitologie Biomédicale and CNRS URA 2581, Institut Pasteur, 75724 Paris Cedex 15, France4

Received 2 July 2003/ Returned for modification 7 September 2003/ Accepted 24 November 2003

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites—Plasmodium falciparum, P. ovale, and P. vivax—was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/µl for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

* Corresponding author. Mailing address: Department of Laboratory Diagnosis, Section of Microbiology, University of Brescia, Spedali Civili, Piazzale Spedali Civili 1, 25123 Brescia, Italy. Phone: 39-030.3995652. Fax: 39-030.395258. E-mail: manca{at}med.unibs.it.

Journal of Clinical Microbiology, March 2004, p. 1214-1219, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1214-1219.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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