On opportunist infections by Trypanosoma lewisi in humans and its differential diagnosis from T. cruzi and T. rangeli.

Trypanosoma lewisi is a cosmopolitan species originally found in Rattus spp., being nonpathogenic, host-restricted, and transmitted by rat fleas. This species has been recorded as an opportunist blood parasite of human beings mainly in Asia, with a case in Africa. In Brazil, this species was recently recorded in captive monkeys. As T. lewisi can share vertebrate hosts both with Trypanosoma rangeli and Trypanosoma cruzi, some markers for the differential diagnosis of these species were examined and discussed herein. The identification of T. lewisi was based on morphological features of bloodstream stages at the initial phase of infection in mammals, isoenzyme electrophoresis at the MDH locus, and PCR products of kinetoplast DNA (kDNA) minicircles using the primers TC121/TC122.

Widefield microscopy for live imaging of lipid domains and membrane dynamics.

Within the lateral organisation of plasma membranes of polarized cell types there exist heterogeneous microdomains of distinct lipid composition, the small size of which (10-200 nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes.