Isolation, propagation and characterization of "Trypanosoma brucei gambiense" from human African trypanosomosis patients in south Sudan

Abstract

Sleeping sickness or Human African trypanosomosis (HAT) is a protozoal disease that is transmitted by tsetse fly vectors in Africa. Sleeping sickness due to T. b. gambiense is a major public health problem in countries in central and western Africa including Angola, Uganda, Democratic Republic of Congo (DRC) and Sudan. High rates of relapses (>20%) following melarsoprol treatment have been reported in many treatment centres including the MSF-F treatment centre at Ibba in south Sudan. The treatment failures could be due to individual (patients) variation in the drug pharmacokinetics, the patient’s immune responses, or drug resistant parasites. There is a growing interest in the elucidation of the reason(s) for relapses after melarsoprol treatment. Since the drug levels in blood or CSF do not differ between relapse and successfully treated patients, drug resistance has been suggested as a likely cause for melarsoprol treatment failures. There are no recently isolated parasites from high-relapse areas and hence detailed studies have been hindered. The objective of this PhD study was to isolate and characterize (phenotypically and genotypically) T. b. gambiense from HAT patients in the MSF-F treatment centre at Ibba. In a first step, the protocols for the isolation of bloodstream forms of the parasite needed to be improved. As such, the suitability of the commercial cryomedium TriladylO developed for bull semen was evaluated. We found that, the cryopreservation of T. b. gambiense in this medium led to a better survival of the trypanosomes than in the standard 10% glycerol. The samples (blood (50) and CSF (2)) from HAT patients were therefore cryopreserved using Triladyl® and stored at -150oC in nitrogen vapour in a dry shipper. In the laboratory, attempts to propagate the isolated parasites in rodents were carried out. Of the 42 parasite positive isolates, 18 (43%) could be propagated in laboratory rodents (immunosuppressed Mastomys natalensis and SCID mice). Stabilates of these T. b. gambiense isolates are stored in two cryobanks at the Trypanosomiasis Research Centre (TRC) of the Kenya Agricultural Research Institute (KARI), Nairobi and the Swiss Tropical Institute (STI), Basel. After the initial isolation of these T. b. gambiense isolates in immunosuppressed M. natalensis or SCID mice, further in vivo propagation could be done in various immunosuppressed rodent species (Swiss White mice, M. natalensis, C57/bl, C3H, and BALB/C). The highest parasitaemia were achieved in C57/bl and BALB/C mice. The rodents however had to be immunosuppressed with cyclophosphamide at 300mg/kg prior to infection and repeated once a week at 200mg/kg. This scheme (cryopreservation and subsequent propagation) allows the isolation of T. b. gambiense from various endemic areas and therefore enhances monitoring of drug resistant trypanosomes. The eighteen T. b. gambiense isolates were found to be sensitive to melarsoprol, melarsen oxide, and diminazene. The gene that codes for the P2 transporter, TbATI, was amplified by PCR and sequenced. The sequences were almost identical to the TbAT1sensitive reference, except for one point mutation, C1384T resulting in the amino acid change proline-462 to serine. None of the described TbAT1resistant-type mutations were detected. In summary we found, in a sleeping sickness focus where melarsoprol had to be abandoned due to the high incidence of treatment failures, no evidence for drug resistant trypanosomes or for TbAT1resistant-type alleles of the P2 transporter. In conclusion, our findings cast doubts on the current suggestion that melarsoprol resistant parasites are the cause of the high rate of treatment failures reported. However, it is important to note that the number of isolates tested was still small and it is vital that more isolates, especially from relapse patients be tested