Year: 2010
Type: article
Abstract: Malaria is a major global health problem, responsible for several hundred million debilitating acute infectious episodes and killing an estimated 800,000 people annually, most of them children under 5 years of age and many of the rest pregnant women. Diagnosis and management of malaria and determination of efficacy of drugs, vaccines, and other interventions all depend on accurate detection of the Plasmodium parasites that cause the disease, determination of which species is/are involved, and, often, quantification of parasite density, expressed as the number of parasites/μL of blood or as the fraction or percentage of parasitized erythrocytes. The mainstay of diagnosis since the late 1800's has been microscopy, using Giemsa's and similar stains to detect parasites in blood smears, but results are limited in accuracy and precision by inadequate training of observers and by the relatively small amount of sample they can practically examine. Both automated microscopy and flow cytometry have been found effective for parasite detection, but require apparatus too expensive and complex to be usable for diagnosis in the resource-poor areas most affected by malaria. Flow cytometry has, however, been utilized in malaria research since the 1970's (1), and newer cytometric applications, such as the paper by Pattanapanyasat et al. published in this issue reflect an increasing level of sophistication (Pattanapanyasat K, Sratongno P, Chimma P, Chitjamnongchai S, Polsrila K, Chotivanich K. Febrile temperature but not proinflammatory cytokines promotes phosphatidylserine expression on Plasmodium falciparum malaria-infected red blood cells during parasite maturation. Cytometry A 2010; in press. DOI: 10.1002/cyto.a.20879). These authors used fluorescein-labeled annexin to study the effects of P. falciparum infection on phosphatidylserine expression of erythrocytes, demonstrating that cell surface phosphatidylserine expression increases with parasite maturation (measured by nucleic acid content detected with ethidium produced by intracellular hydroethidine oxidation) and is also influenced by fever, infection rate, and the parasite strain. This work is particularly important as it provides evidence that P. falciparum causes asymmetry of membrane phospholipids and is crucial for senescence of uninfected erythrocytes, whereas proinflammatory cytokine action is not important for erythrocyte senescence. Giemsa's stain binds to both DNA and RNA, but cannot be used for quantification of either. Flow cytometric analyses of malaria began in the 1970's (1). Most early studies utilized dyes, such as acridine orange, ethidium, and propidium, which are readily excited by 488 nm lasers, the only illumination then available on most instruments. These dyes, and the newer SYTO and YOYO series, typically stain DNA and RNA indistinguishably, and most require that cells be fixed or permeabilized. The few investigators who did have early access to the ultraviolet (UV) sources needed to excite DNA-specific, stoichiometric stains were likely to use Hoechst 33258 or DAPI, which require permeabilization, rather than Hoechst 33342, which does not. Plate reader DNA content measurements of cultured plasmodia in bulk have been used to screen antimalarial drugs; the sensitivity of such assays is improved if imaging of wells or flow cytometry is used to detect and quantify DNA in individual parasites (2, 3). Modern flow cytometers typically have at least two laser sources, and a configuration with violet or UV, red, and 488 nm beams is now fairly common. Those who can afford such apparatus can measure six or more parameters, and reagents now available permit a wide range of multiparameter analyses to be done on unfixed cells. In 1986, Hare and Bahler (4) used two-color flow cytometric acridine orange fluorescence measurements and cell sorting to show that quantification of DNA and RNA precisely identified intraerythrocytic developmental stages of malaria parasites without the need for any morphologic information. The finding went largely unnoticed until 2008, when Grimberg et al. (5) combined Hoechst 33342 and thiazole orange to quantify DNA and RNA in a flow cytometric screen for antimalarials; they more recently added a measurement of mitochondrial membrane potential using the red-excited cyanine dye DiIC1 (5) to determine parasite viability (6), further refining the screening procedure. In recent years, a number of visible-excited dyes for DNA quantification in unfixed cells, for example, SYBR Green (3), DRAQ5, and the VybrantDyeCycle series have become available; this enables DNA and DNA/RNA staining to be done in instruments lacking the UV or violet lasers necessary to excite Hoechst 33342 and related dyes. Cells' content of hemozoin (7), which maturing plasmodia produce by breaking down hemoglobin, would be an essential measurement parameter in a “malaria dream machine” cytometer. Hemozoin content of parasitized cells can respond rapidly to drugs to which the parasites are susceptible, and phagocytosed hemozoin in neutrophil granulocytes and monocytes may provide an indicator of severity of infection, especially in children. Hemozoin is birefringent and easily measurable by intensities of scatter and/or transmission of light of orthogonal polarizations; however, a patent licensed to Abbott Diagnostics and utilized in their hematology analyzers has, until its recent expiration, inhibited manufacturers of fluorescence flow cytometers from incorporating the measurement capability. Cytometric methods have also been used for measuring oxidative stress in P. falciparum-infected erythrocytes (8), melatonin-induced calcium fluxes underlying maturation of P. falciparum (9) and parasite-protein trafficking [reviewed in (10)]. A number of antimalarials affect metabolism of glutathione, but established cytometric methods for measurement of it and of other thiols have apparently not yet been applied to studies of plasmodia. Although some enthusiasm for increased use of flow cytometry in malaria diagnosis and management has recently been expressed (11), we believe it is unlikely that the cost and complexity of flow cytometers will decrease sufficiently to get the apparatus everywhere it is needed. As has, however, been pointed out previously (12), many multiparameter optical measurements now made by flow cytometers can also be made in much simpler and cheaper instruments, which image a large enough field to eliminate the need for stage motion and focus adjustment, while rapidly examining enough of a blood specimen to derive a more precise estimate of parasite density than could practically be obtained by microscopy. Such imagers could be used for diagnosis of malaria, and should also permit a wider range of clinical and experimental cytometric studies to be done in resource-poor areas. Most fatal malaria is caused by Plasmodium falciparum; it is therefore a priority in diagnosis to distinguish pure and mixed infections involving this species from those due to P. vivax, P. malariae, P. ovale, and P. knowlesi, all less likely to cause life-threatening illness. P. falciparum DNA contains 80% adenine plus thymine, whereas these bases only comprise about 60 per cent of the DNA of humans and of the other human malaria parasites. Combinations of DNA dyes with different base pair preferences can easily detect differences of this magnitude, having been used successfully for analyses of chromosomes and bacteria. Additional measurements of RNA content and hemozoin, which, like DNA measurements, yield relatively high-intensity optical signals, might be sufficient to discriminate P. falciparum from the other species that infect man. Configuring a practical diagnostic instrument will, however, require collection of good cytometric data on the three nonculturable species of human malaria parasite, P. vivax, P. malariae, and P. ovale, as there are only a handful of papers on cytometry of the first of these and none of which we are aware on the other two. This may ultimately necessitate taking a suitably ruggedized cytometer where the patients are. We hope this will be done within the next year and expect that the same measurements will by then be incorporated into an inexpensive system for antimalarial screening and other research applications requiring discrimination of developmental stages but not of species.
Source: Cytometry Part A
DOI: 10.1002/cyto.a.20903
Authors: Howard M. Shapiro, Henning Ulrich
Institution: Universidade de São Paulo
Language: English
Topic: Malaria Research and Control
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2079487779