A colorimetric assay based on cell viability for the indirect detection of intracellular replication and killing of Trypanosoma cruzi.

We have developed an 'in vitro' colorimetric assay for the detection of replication and/or destruction of the intracellular amastigote form of Trypanosoma cruzi. The assay can be applied to other intracellular parasites having lytic effects on the infected cells. The assay is based on the relationship between the content of the lysosomal enzyme hexosaminidase and the number of viable cells. The level of this enzyme can be detected by a simple and sensitive procedure in microtiter wells using a p-nitrophenol derivative as enzyme substrate and scanning the absorbance at 405 nm. Macrophages or other suitable host cells which support T. cruzi intracellular replication have detectable levels of this enzyme whereas the protozoan parasites do not. The assay exhibited a good inverse correlation between the number of amastigotes released and the amount of enzyme in the infected cultures. Furthermore, the method was used for the detection of macrophage-activating factors and gave results similar to those obtained by microscopical examination of the cells. The advantages of the procedure are objectivity, sensitivity and simplicity.