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We studied the accuracy of PFR1-AP, a synthetic DNA hybridization probe conjugated to alkaline phosphatase, in monitoring Plasmodium falciparum parasitemia during in vivo drug susceptibility surveys. Duplicate blood samples were collected from six children enrolled in a 14-day in vivo chloroquine study in Rwanda. Results obtained by microscopic examination of Giemsa-stained thick blood smears and by DNA hybridization were compared. Both techniques successfully monitored an infection with chloroquine-susceptible parasites and infections exhibiting various levels of resistance to treatment. For each patient, temporal evolution of the microscopic parasite counts and the DNA hybridization signals were closely parallel, although a wide range of rapidly changing levels of parasitemia occurred through the course of the study. This suggests that DNA hybridization assay using PFR1-AP detects P. falciparum parasites sensitively and specifically and is a valuable tool for drug resistance surveys.

Total genomic Plasmodium falciparum DNA, the plasmid clone pRepHind, and a 21-base-long synthetic DNA probe (PFR1), the sequence of which was derived from pRepHind, were hybridized with DNA from various species of the phylum Apicomplexa. The genomic probe hybridized with P. reichenowi and P. falciparum DNA and significantly cross-hybridized with DNA of all the other Plasmodium species tested. The synthetic and plasmid probes hybridized to P. falciparum DNA and at reduced levels to P. reichenowi but did not hybridize to P. vivax, P. malariae, P. ovale, P. fragile, P. inui, P. knowlesi, Babesia bovis, B. microti, B. bigemina, Anopheles sp., Pan sp., Aotus sp., or human DNA. Southern blot analysis indicated that approximately 60 distinct restriction enzyme fragments from P. falciparum DNA were similarly detected by PFR1 and pRepHind. A method was developed by using a second brief hybridization with synthetic DNA to amplify signals from samples that were previously hybridized with plasmid-borne repetitive DNA. This amplification procedure was shown to allow the detection of 0.005% P. falciparum parasitemias from 10-microliter samples of blood from patients in Kenya.