Isolation of Trypanosoma brucei gambiense from Cured and Relapsed Sleeping Sickness Patients and Adaptation to Laboratory Mice

Abstract

BackgroundSleeping sickness due to Trypanosoma brucei (T.b.) gambiense is still a major public health problem in some central African countries. Historically, relapse rates around 5% have been observed for treatment with melarsoprol, widely used to treat second stage patients. Later, relapse rates of up to 50% have been recorded in some isolated foci in Angola, Sudan, Uganda and Democratic Republic of the Congo (DRC). Previous investigations are not conclusive on whether decreased sensitivity to melarsoprol is responsible for these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as from relapsed patients for downstream comparative drug sensitivity profiling. A major constraint for this type of investigation is that T.b. gambiense is particularly difficult to isolate and adapt to classical laboratory rodents.Methodology/Principal FindingsFrom 360 patients treated in Dipumba hospital, Mbuji-Mayi, D.R. Congo, blood and cerebrospinal fluid (CSF) was collected before treatment. From patients relapsing during the 24 months follow-up, the same specimens were collected. Specimens with confirmed parasite presence were frozen in liquid nitrogen in a mixture of Triladyl, egg yolk and phosphate buffered glucose solution. Isolation was achieved by inoculation of the cryopreserved specimens in Grammomys surdaster, Mastomys natalensis and SCID mice. Thus, 85 strains were isolated from blood and CSF of 55 patients. Isolation success was highest in Grammomys surdaster. Forty strains were adapted to mice. From 12 patients, matched strains were isolated before treatment and after relapse. All strains belong to T.b. gambiense type I.Conclusions and SignificanceWe established a unique collection of T.b. gambiense from cured and relapsed patients, isolated in the same disease focus and within a limited period. This collection is available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, D.R. Congo.

Pharmacokinetics, Trypanosoma brucei gambiense Efficacy, and Time of Drug Action of DB829, a Preclinical Candidate for Treatment of Second-Stage Human African Trypanosomiasis

“…The first relapse was detected 1 year after a 3-day melarsoprol treatment, and the second relapse was detected 8 months after a melarsoprol-nifurtimox combination therapy (29). Strain 40R was also isolated from a patient in DRC in 2005, 6 months after failure of the 10-day melarsoprol treatment (29). The strain 349R was isolated from a patient in DRC in 2006 after a treatment failure with a 10-day melarsoprol treatment (29).…”

“…Strain 40R was also isolated from a patient in DRC in 2005, 6 months after failure of the 10-day melarsoprol treatment (29). The strain 349R was isolated from a patient in DRC in 2006 after a treatment failure with a 10-day melarsoprol treatment (29). DAL898R was isolated from a patient in Cote d'Ivoire in 1985 after melarsoprol treatment failure (23).…”

“…Strain 130R was isolated from a patient in DRC in 2005 after two treatment failures with melarsoprol. The first relapse was detected 8 months after a 3-day melarsoprol treatment, and the second relapse was detected 1 year after a melarsoprol-nifurtimox combination therapy (29). Strain 45R was isolated from a patient in DRC in 2005 after two treatment failures with melarsoprol.…”

Luminescent multiplex viability assay for Trypanosoma brucei gambiense

“…rhodesiense and T.b. gambiense strains that are recently isolated from patients with known treatment outcomes and that underwent few in vivo and in vitro passages, should be included in these drug discovery validation panels [21-23]. It would be even better to include T.b.…”

Antitrypanosomal Activity of Fexinidazole, a New Oral Nitroimidazole Drug Candidate for Treatment of Sleeping Sickness

“…b. gambiense strains 40R, 45R, 130R, 349Pi, and 349R were all isolated from patients in the Democratic Republic of Congo in 2003 to 2004 (26). The K03048 strain was isolated from a patient in South Sudan in 2003 (20).…”