Abstract
BackgroundNew compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient viability assay to monitor drug efficacy.MethodsIn this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex viability assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin viability assay for drug sensitivity testing of T.b. gambiense.ResultsIn a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 104 cells ml-1 for the Renilla luciferase measurement and 5 × 103 cells ml-1 for the ATP measurement. Both assays of the LMVA showed linearity up to 106 cells ml-1 and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin viability assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both viability assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both assays of the LMVA.ConclusionsLMVA has some advantages for viability measurement of T.b. gambiense: it requires less incubation time for viability detection than the fluorimetric resazurin assay and in LMVA, two sensitive and independent viability assays are performed in the same experiment.
Highlights
New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required
It has been shown that the EnduRen assay, for measurement of vital in vitro Renilla luciferase activity, can be combined with the CellTiter-Glo assay as an efficient luminescent multiplex viability assay for highthroughput screenings (HTS) compound screening against Dengue [42]
In vitro adaptation in HMIhuman serum (HH) To be compatible with compound screening, it was necessary to adapt the T.b. gambiense 348BT strain that readily grows in HMI – human serum – methylcellulose (HHM) to a medium without methylcellulose because the latter is very viscous, which renders homogenous volume distribution and centrifugation very difficult
Summary
New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. The reason for low conversion of resazurin to resorufin is unknown, but long term incubation times with resazurin with live or lysed trypanosomes may affect the IC50 value of a drug [26,31,32] Because of their easy, sensitive and fast readout, viability assays based on ATP detection (such as the luminescent CellTiter-Glo assay) have been proposed and used as an alternative viability assay for HTS in T.b. brucei strain 427 [5,7]. A T.b. gambiense strain that was recently isolated in the Democratic Republic of the Congo was made bioluminescent by expression of Renilla luciferase With this strain, the feasibility of the Enduren/CellTiter-Glo luminescent multiplex viability assay, abbreviated as LMVA, was compared to the fluorimetric resazurin assay for drug sensitivity testing of the main drugs to treat T.b. gambiense sleeping sickness: pentamidine, eflornithine nifurtimox and melarsoprol
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