Feasibility of Flow Cytometry for Measurements of Plasmodium falciparum Parasite Burden in Studies in Areas of Malaria Endemicity by Use of Bidimensional Assessment of YOYO-1 and Autofluorescence

The detection and quantification of Plasmodium falciparum in studies of malaria endemicity primarily relies upon microscopy. High-throughput quantitative methods with less subjectivity and greater reliability are needed for investigational studies. The staining of parasitized erythrocytes with YOYO-1 for flow cytometry bears great potential as a tool for assessing malaria parasite burden. Capillary blood was collected from children presenting to the pediatric ward of the Manhic, a District Hospital in Mozambique for parasitemia assessment by thick and thin blood films, flow cytometry (YOYO-1(530/585)), and quantitative real-time PCR (qRT-PCR). Whole blood was fixed and stained with YOYO-1 for acquisition on a cytometer to assess the frequency of infected erythrocyte events. qRT-PCR was used as the gold standard for the detection of P. falciparum. The YOYO-1(530/585) method was as sensitive and specific as conventional microscopy (area under the receiver operating characteristic, 0.9 for both methods). The interrater mean difference for YOYO-1(530/585) was near zero. Parasite density using flow cytometry and complete blood counts returned density estimates with a mean difference 2.2 times greater than results by microscopy (confidence interval, 1.46 to 3.60) but with limits of agreement between 10 times lower and 50 times higher than those of microscopy. The YOYO-1(530/585) staining pattern was established exactly as demonstrated in animal models, but the assay was limited by the lack of appropriate negative-control samples for establishing background levels and the definition of positives in areas in which malaria is endemic. YOYO-1(530/585) is a high-throughput tool with great potential if the limitations of negative controls and heterogeneous levels of background signal can be overcome.