Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings.

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Four Plasmodium species cause malaria in humans. Most malaria-endemic regions feature mixed infections involving two or more of these species. Factors contributing to heterogeneous parasite species and disease distribution include differences in genetic polymorphisms underlying parasite drug resistance and host susceptibility, mosquito vector ecology and transmission seasonality. It is suggested that unknown factors limit mixed Plasmodium species infections, and that mixedspecies infections protect against severe Plasmodium falciparum malaria. Careful examination of methods used to detect these parasites and interpretation of individual-and population-based data are necessary to understand the influence of mixed Plasmodium species infections on malarial disease. This should ensure that deployment of future antimalarial vaccines and drugs will be conducted in a safe and timely manner. Although novel exceptions have been reported [1], it is commonly agreed that Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale are the four species that cause human malaria. Factors underlying mixed infections involving these parasite species have been discussed since 1930, when Knowles and White acknowledged difficulties that microscopists might encounter in accurately documenting their findings from examinations of blood smears [2]. Humans often harbor multiple Plasmodium species [3][4][5][6], and varying patterns in species-specific parasitemia and mixed-species prevalence characterize malaria infections in different endemic regions ([7-10]; reviewed in Refs [11,12]). Data from malaria fever therapy patients involving different Plasmodium species [13][14][15], and naturally infected study volunteers, have illustrated a range of observations from orderly to turbulent species-specific patterns (Figure 1) of parasitemia in infected individuals [13][14][15][16][17]. Antimalarial treatment studies have also contributed insight regarding mixed Plasmodium species infections by revealing undocumented infection of a second species following successful treatment of a first species (reviewed in Ref. [18]). These varied patterns of parasitemia observed in individuals infected by multiple Plasmodium species are surely influenced by a complex array of host factors [19] acting to constrain the infection before the parasites completely over-run the available erythrocyte population. Evidence that the four Plasmodium parasites of humans antagonize one another seems less clear.Conflicting results suggesting the presence or absence of mixed-species interactions from one study to the next are seldom resolved. If direct interaction between Plasmodium species occurs within an infected individual to any significant level, these interactions would then be expected to influence the distribution of species within the endemic population. A common theme discussed in the context of Plasmodium species interactions has focused on