RESUMEN
BACKGROUND: Sleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba. METHODOLOGY/PRINCIPAL FINDINGS: Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1. CONCLUSIONS/SIGNIFICANCE: We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.
Asunto(s)
Acuagliceroporinas/genética , Melarsoprol/farmacología , Tripanocidas/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Tripanosomiasis Africana/parasitología , Adulto , Animales , Secuencia de Bases , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , República Democrática del Congo , Resistencia a Medicamentos/genética , Femenino , Genotipo , Humanos , Melarsoprol/uso terapéutico , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/genética , Mutación , Pentamidina/farmacología , Fenotipo , Recurrencia , Análisis de Secuencia de ADN , Tripanocidas/uso terapéutico , Trypanosoma brucei gambiense/genética , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
BACKGROUND: Sleeping 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 FINDINGS: From 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 SIGNIFICANCE: We 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.
