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

Abstract

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 Manhiça 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.

Malaria diagnosis for malaria elimination

“…Further, hemozoin-containing monocytes observed by microscopy may provide insight regarding the total body parasite burden, as these cells do not sequester in P. falciparum infections [49,50]. Flow cytometric devices may also contribute new approaches for malaria diagnosis with emphasis on field deployment, however, further development of instrumentation is required [51][52][53][54][55]; limitations of speciesspecific detection must be acknowledged for flow cytometry.…”

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

“…A comparison between microscopic analysis following Giemsa staining and FCM shows a very similar distribution (Fig. 1D) (14,23,24), YOYO-1 presented an excellent discrimination between uninfected and infected RBCs and was able to discriminate between mononucleated and multinucleated stages with high resolution. The fluorescence intensities of multinucleated-stage parasites (Fig.…”

“…Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia in cultures of P. falciparum by FCM. Some previous studies using YOYO-1 showed that this dye possesses high affinity for DNA and discriminates infected RBC from the uninfected cells in a very effective way (14,23,24). Melatonin and derivatives mediating effects on the cell cycle of P. falciparum-infected human erythrocytes were studied using YOYO-1 staining followed by flow cytometry analysis.…”

Light depolarization measurements in malaria: A new job for an old friend

“…Cytometry of malaria-infected RBC has been used for (i) monitoring of infected RBC, mainly in mouse models (34)(35)(36)(37)(38)(39), (ii) characterization of infected RBC (15), (iii) assessment of maturation and/or viability of parasites in infected RBC (10,13,14,16,17,40,41), and (iv) to detect and count P. falciparum infected RBC in culture (14,(42)(43)(44) or from patient blood (45)(46)(47), with the possibility to distinguish species based on base composition (2,10,11).…”