Only viable parasites are detected by PCR following clearance of rodent malarial infections by drug treatment or immune responses

Abstract

Detection and analysis of pathogens by PCR plays an important role in infectious disease research. The value of these studies would be diminished if nuclear material from dead parasites were found to remain in circulation for extended periods and thus result in positive amplification. This possibility was tested in experimental rodent malaria infections. Blood samples were obtained from infected mice during and following drug or immune clearance of Plasmodium chabaudi chabaudi parasitemias. Detection of parasite DNA by a sensitive Plasmodium-specific PCR amplification assay was associated with the presence of viable parasites, as detected by subinoculation. No parasite DNA could be detected by PCR 48 h after the injection of killed parasites into mice. Nuclear material from parasites removed by drug or immune responses is rapidly cleared from the circulation and does not contribute significantly to amplification. Thus, results from PCR analysis of malaria-infected blood accurately reflect the presence of live parasites.

FIG. 1

Following the addition of 5 μl of loading buffer, 15 μl of the PCR product was electrophoresed on a 3% MetaPhor agarose gel (Flowgen Instruments Ltd.) in TBE buffer (100 mM Tris-HCl, 100 mM boric acid, 5 mM EDTA) and visualized by UV transillumination after ethidium bromide staining. Molecular weight markers (lanes M) are a 100-bp ladder; the size of the lowest band visible is 100 bp. Results (+ or −) of subinoculations are given for reporter mice below each gel. The gels and the subinoculation results are aligned under the corresponding day when the sample analyzed was collected. (A) PCR and subinoculation analyses of blood samples obtained after injection of freeze-thawed P. c. chabaudi AS parasites into a representative mouse. The sample taken from the mouse before injection of parasite material is designated B, and the one taken immediately after is A. Values above the gel are the numbers of days following the injection of parasite material. (B) Parasitemia curve and PCR-subinoculation analyses of a representative mouse in which the parasitemia was cleared by pyrimethamine treatment (initiated on day 10, as indicated by the arrow designated T). (C) Parasitemia curve and PCR-subinoculation analyses of parasite clearance in a representative hyperimmune mouse. The challenge inoculum was administered immediately after collection of sample B. E, erythrocytes.