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Abstract. We have developed two diagnostic assays based on the specific detection of Plasmodium lactate dehydrogenase (pLDH) activity. These assays exploit a panel of monoclonal antibodies that capture the parasite enzyme and allow for the quantitation and speciation of human malaria infections. An immunocapture pLDH activity assay (ICpLDH) allows for the rapid purification and measurement of pLDH from infected blood using the NAD analog APAD, which reacts specifically with Plasmodium LDH isoforms. An immunochromatographic test (the OptiMAL assay) was also formatted and allowed the detection of parasite infections of ϳ200 parasites/l of blood. By using a combination of antibodies, both tests can not only detect but differentiate between P. falciparum and non-P. falciparum malaria. Both assays show a sensitivity comparable with other commercial nonmicroscopic tests; importantly, we found very few instances of false-positive samples, especially with samples from patients recently cleared of malaria infection. Furthermore, we find that when one uses the quantitative ICpLDH assay, the levels of pLDH activity closely mirror the levels of parasitemia in both initial diagnosis and while following patient therapy. We conclude that diagnostic tests based on the detection of pLDH are both sensitive and practical for the detection, speciation, and quantitation of all human Plasmodium infections and can also be used to indicate drug-resistant infections.

In 1891 Guttmann and Ehrlich (P. Guttmann and P. Ehrlich, Berlin Klin. Wochenschr. 28:953-956, 1891) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. Since that time, sporadic reports of the antimalarial properties of several xanthene and azine dyes related to methylene blue have been noted. We report here the results from a reexamination of the antimalarial properties of methylene blue, Janus green B, and three rhodamine dyes and disclose new antimalarial data for 16 commercially available structural analogs of these dyes. The 50% inhibitory concentrations for the chloroquinesusceptible D6 clone and SN isolate and the chloroquine-resistant W2 clone of Plasmodium falciparum were determined by the recently described parasite lactate dehydrogenase enzyme assay. No cross-resistance to chloroquine was observed for any of the dyes. For the 21 dyes tested, no correlation was observed between antimalarial activity and cytotoxicity against KB cells. No correlation between log P (where P is the octanol/ water partition coefficient) or relative catalyst efficiency for glucose oxidation and antimalarial activity or cytotoxicity was observed for the dyes as a whole or for the thiazine dyes. The thiazine dyes were the most uniformly potent structural class tested, and among the dyes in this class, methylene blue was notable for both its high antimalarial potency and selectivity.In 1891 Guttmann and Ehrlich (25) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. The only side effect that they noted was a ''spastic irritation of the bladder,'' which was partially alleviated by the administration of powdered nutmeg. They selected methylene blue because it had been previously demonstrated by Celli and Guarnieri to specifically stain plasmodia (25) and also because Ehrlich and Leppmann had prior experience in the therapeutic use of this dye in the successful clinical treatment of neuralgias (32). Guttmann and Ehrlich (25) apparently made no further attempt to pursue this interesting observation, although as late as 1956, Dale (14), in his introduction to The Collected Papers of Paul Ehrlich, noted that ''methylene blue itself has there [in the Balkan countries] retained a fairly extensive use, for the treatment of malaria in cases which have been found refractory to quinine.'' Moreover, methylene blue, along with quinine, served as structural prototypes (48) which led to the subsequent development of the 8-aminoquinoline pamaquine and the 9-aminoacridine mepacrine (quinacrine) in 1925 and 1930, respectively (Fig. 1).In the past decade, sporadic reports (7,18,19,21,27) of the antimalarial properties of several xanthene (rhodamines 123, 6G, and B) and azine (Janus green B) dyes related to methylene blue ha...

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