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.
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.
Machouart
M
M
Laboratoire de Parasitologie-Mycologie, CHU Brabois, 54500 Vandoeuvre-les-Nancy, France. m.machouart@chu-nancy.fr
Bigois-Delemotte
L
L
Ajana
F
F
Brizion
M
M
Biava
M F
MF
Collomb
J
J
Fortier
B
B
D000818:Animals; D001483:Base Sequence; D001769:Blood / Q000469:parasitology*; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D018512:Parasitemia / Q000175:diagnosis / Q000469:parasitology; D010961:Plasmodium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S