A rapid in vitro test system has shown that many drugs which possess a product licensed for use in man are also active at a concentration of less than 1mM against the blood forms of Trypanosoma cruzi. 62 of these are structurally related amphiphilic cationic drugs which completely lyse the trypomastigotes at 4 degrees C within 24 hr, yet most leave the erythrocytes intact. Three polyene and two anthracycline antibiotics were also found to be selectively trypanocidal under the same conditions.

SummaryIsolation of blood and intracellular forms of Trypanosoma cruzi was made mainly from rats (90–110 g) which had received 580 rad of whole-body γ-irradiation not more than 24 h before subcutaneous inoculation with 107 trypomastigotes of the Sonya strain of T. cruzi. Unirradiated chinchillas (250–350 g) were, however, used for some experiments. Blood forms were isolated using a technique involving differential centrifugation to remove most of the erythrocytes and DEAE–cellulose chromatography to remove the remaining blood cells. Overall recoveries were usually in the range 30–70%. Parasites were mainly (approximately 98%) broad forms and were motile, metabolically active (as judged by respiratory and radio-tracer incorporation studies) and had lost none of their infectivity for mice. Intracellular forms were isolated from hind-limb muscle tissue. This was disrupted in an MSE tissue homogenizer and the homogenate incubated with DNase, collagenase and trypsin. Parasites, contaminated only by a few blood cells, were then obtained by differential centrifugation. For purer preparations, a terminal sucrose gradient step was used. Recoveries ranged between 40 and 70%. About 1–3% of the parasites isolated were epimastigotes and trypomastigotes; the remainder are probably best collectively termed ‘amastigotes’, though they were pointed and most had a short, free flagellum. They were undamaged as judged by light and electron microscopy and metabolically active as judged by respiratory and radio-tracer incorporation studies. However, the infectivity for mice of both these purified preparations and the initial cell homogenates could be accounted for by the epimastigotes and trypomastigotes present in them. Preliminary biochemical studies with isolated parasites have shown that blood, intracellular and culture forms of T. cruzi have a respiratory system which is in part sensitive to CN- and that all forms synthesize nucleic acids and proteins when incubated in vitro. There appears, however, to be a lack of DNA synthesis in blood stages, and thus it is not surprising that these forms do not divide.

Addition of beta-lapachone, an o-naphthoquinone with bactericidal, cytotoxic, and trypanocidal activities, to Trypanosoma cruzi epimastigote and amastigote stages induced the release of O2- and H2O2 from the whole cells into the suspending medium. In the presence of reduced nicotinamide adenine dinucleotide as reductant beta-lapachone was also able to stimulate O2- and H2O2 production by homogenates of these stages. Electron micrographs showed that in beta-lapachone-treated amastigotes and trypomastigotes, the chromatin is arranged in patches, clearly differing from the normal pattern of chromatin distribution. Alterations of the nuclear, mitochondrial, and cytoplasmic membranes, as well as swelling of the mitochondria were also observed.

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This paper describes how Microslide tubes can be used as the basis for a quick, simple, inexpensive screen to test for compounds which might prevent the transmission of Chagas's disease as a result of blood transfusion. The test uses Trypanosoma cruzi-infected mouse blood and requires only very small amounts of compound. The screen identifies compounds known to be active against blood trypomastigotes in vitro at 4 degrees C, and is apparently free of false negative and positive results.