title: Luminescent multiplex viability assay for Trypanosoma brucei gambiense.
abstract: BACKGROUND
  New 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.
  
  
  METHODS
  In 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.
  
  
  RESULTS
  In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 10(4) cells ml(-1) for the Renilla luciferase measurement and 5 × 10(3) cells ml(-1) for the ATP measurement. Both assays of the LMVA showed linearity up to 10(6) 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.
  
  
  CONCLUSIONS
  LMVA 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.
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium. nvanreet@itg.be
authors.forename: Nick
authors.initials: N
authors.lastname: Van Reet
authors.forename: Pati
authors.initials: P
authors.lastname: Pyana
authors.forename: Stijn
authors.initials: S
authors.lastname: Rogé
authors.forename: Filip
authors.initials: F
authors.lastname: Claes
authors.forename: Philippe
authors.initials: P
authors.lastname: Büscher
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002470:Cell Survival; D020128:Inhibitory Concentration 50; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010274:Parasitology / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D014347:Trypanosoma brucei gambiense / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*
chemical_list: D000981:Antiprotozoal Agents; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D049410:Luciferases, Renilla