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A cell viability assay, using fluorescence measurements has been developed for the screening of new compounds against African trypanosomes. 2',7'-Bis-(carboxyethyl)-5(6)-carboxyfluorescein-pentaacetoxymethyles ter (BCECF-AM), an esterase substrate, was used in the assay as a marker for cell viability. Fluorescence was quantified using an automated fluorescence scanner for multi-well plates. Trypanosoma brucei rhodesiense, T. congolense, T. evansi and T. equiperdum from continuously growing cultures were exposed to various concentrations of trypanocidal drugs for an incubation period of 72 h at 37 degrees C. Then BCECF-AM was added to the cell suspensions and after 60 minutes the fluorescence of the trypanosome suspension was measured using the Millipore Cytofluor 2300 fluorescence scanner, at 485 nm excitation and 530 nm emission wavelengths. Results of kinetic studies of the hydrolysis of the non-fluorescent BCECF-AM in trypanosomes showed that BCECF-AM is readily cleaved by non-specific esterases to a highly fluorescent product. Drug concentrations causing 50% inhibition of fluorescence (IC50-values) were measured fluorimetrically. Minimum inhibitory concentration (MIC) was determined microscopically.

Thirty eight Trypanosoma brucei rhodesiense-infected vervet monkeys (Cercopithecus aethiops) in the late (meningoencephalitic) stage of disease, treated with various trypanocidal drugs, were monitored for a period of more than 600 days to assess the rate of clearance of trypanosome antigens from serum and cerebrospinal fluid (CSF). There was a complete but gradual reduction in antigen titres, as assessed by ELISA, in animals treated intravenously with melarsoprol, the standard drug for the late stage disease. In 8 of the 9 monkeys treated with melarsoprol, the antigen titres, as assessed by optical density values, dropped by 50% within 252 days (mean value 68 days for antigens in CSF and 116 for serum) following treatment. The remaining animal in this group, that displayed persistent antigenaemia, had been treated with a sub-curative drug dosage level. Thus, if time to 50% reduction in antigen levels were to be taken as an index to predict cure, the follow-up period after melarsoprol treatment could have been reduced from 600 to 252 days for 8 of the 9 animals, leaving only one animal for further follow up. The animals treated with experimental drug combinations displayed a variable picture. Five monkeys showed a persistence of antigens in both serum and CSF throughout the observation period, suggesting failure of the drugs to cure the infection. Parasitologically confirmed relapse of the infection was indeed observed in all the five monkeys. In some monkeys, the parasite antigens eventually cleared from serum and CSF completely, but this took a longer time duration than in the melarsoprol treated animals; others showed persistence of parasite antigens in serum, but the parasites were not detected in blood or CSF throughout the entire follow-up period. These results suggest that the experimental drug combinations used were not effective in clearing the parasites from cryptic foci and hence the persistence of antigens in serum and/or CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to initiate transformation into procyclic forms, bloodstream trypanosomes, were transferred to semi-defined medium at 27 degrees C. All stocks previously classified as Trypanosoma brucei rhodesiense, T.b. gambiense and T.b. brucei transformed into procyclic forms. None of the 31 characterized T. evansi stocks transformed into procyclic forms, but died between day 4 and day 6 in culture. On the other hand, 3 out of 64 stocks of monomorphic brucei subgroup field isolates from camels transformed into procyclic forms, confirming the existence of T.b. brucei infection in camels kept close to tsetse belts.

The RoTat 1/2 CATT test developed for Trypanosoma evansi was used in comparison with other diagnostic tests for the detection of T. rhodesiense infection in the northern part of the Luangwa Valley. The human population, the domestic and a large number of game animals were positive with the RoTat 1/2 CATT, the Ag-ELISA, the IFAT and the radioimmunoprecipitation tests. Human sera from these areas precipitated the same trypanosome-antigen components 35S-methionine labelled whereas few differences in band patterns were found between individual game animals. Surprisingly, however, T. rhodesiense could not be isolated from the "Ag-ELISA and radioimmunoprecipitation" positive patients from the Musenga and Kasyasya localities. The fact that the CATT positive humans were positive in antigen detection tests, does indicate that in all probability they carry or had been carrying a subpatent infection. These results suggest that the reservoir for T. rhodesiense in that region is considerable, comprising the game animals and probably to an even greater extent, the human population.