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Journal of Clinical Microbiology, September 2006, p. 3279-3284, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00014-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

Laboratoire de Parasitologie-Mycologie, CHU Brabois, 54500 Vandoeuvre-les-Nancy, France,¹ Hôpital d'Instruction des Armées Legouest, 57998 Metz, France,² Service Regional Universitaire des Maladies Infectieuses et du Voyageur, Hôpital DRON, 59200 Tourcoing, France³

Received 4 January 2006/ Returned for modification 17 February 2006/ Accepted 15 June 2006

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.