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

Human African trypanosomiasis, or sleeping sickness, is still a major public health problem in central Africa. Melarsoprol is widely used for treatment of patients where the parasite has already reached the brain. In some regions in Angola, Sudan, Uganda and Democratic Republic of the Congo, up to half of the patients cannot be cured with melarsoprol. From previous investigations it is not yet clear what causes these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as relapsed patients for downstream comparative drug sensitivity profiling. From 360 sleeping sickness patients, blood and cerebrospinal fluid (CSF) was collected before treatment and along the prescribed 24 months follow-up. Blood and CSF were inoculated in thicket rats (Grammomys surdaster), Natal multimammate mice (Mastomys natalensis) and immunodeficient laboratory mice (Mus musculus). Thus, we established a unique collection of Trypanosoma brucei gambiense type I parasites, isolated in the same disease focus and within a limited period, including 12 matched strains isolated from the same patient before treatment and after relapse. This collection is now available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, Democratic Republic of the Congo

Schematic view of the <i>AQP2/3</i> variants identified in this study (adapted from Graf et al [20]).

<p>Sequence of <i>AQP3</i> was not verified. Positions of primer: black box = AQP2/3_F, green box = AQP2_R, red box = AQP2/3_R. A) Reference locus of <i>AQP2</i> and <i>AQP3</i>, with wild-type <i>AQP2</i> found in the melarsoprol and pentamidine sensitive strain <i>T.b. gambiense</i> LiTat 1.3 and in all strains from Masi-Manimba. B) Chimera of <i>AQP2</i> and <i>AQP3</i> occurring in a melarsoprol and pentamidine resistant <i>T.b. brucei</i> strain as described by Baker <i>et al.</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003212#pntd.0003212-Baker1" target="_blank">[19]</a>. C) Chimera of <i>AQP2</i> and <i>AQP3</i> plus loss of <i>AQP3</i> in all <i>T.b. gambiense</i> strains from Mbuji-Mayi as described in this article and by Graf et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003212#pntd.0003212-Graf1" target="_blank">[20]</a>. D) New chimera of <i>AQP2</i> and <i>AQP3</i>, possibly outside the known locus, found in all <i>T.b. gambiense</i> strains from Mbuji-Mayi. E) New chimera of <i>AQP2</i> and <i>AQP3</i> plus loss of <i>AQP3</i> found in two old Congolese <i>T.b. gambiense</i> strains, MBA and KEMLO. F) New chimera of <i>AQP2</i> and <i>AQP3</i>, without loss of <i>AQP3</i>, found in all four <i>T.b. gambiense</i> strains isolated in Masi-Manimba.</p

Phenotype of melarsoprol resistant strains.

<p>Number of relapsing mice (out of 6 infected) and day post-infection that relapses were observed after treatment with melarsoprol at different dosages and repetitions. DPI: days post-infection, BW: body weight, rep: repetition, na: not applicable,</p><p>*: relapsing population used for AQP2/3 RFLP analysis.</p><p>Phenotype of melarsoprol resistant strains.</p

Restriction digest profile generated with SfaNI PCR-RFLP on DNA of the <i>T.b. gambiense</i> strains as listed in Table 1 and of the two <i>T.b. brucei</i> control strains.

<p>Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = <i>T.b. gambiense</i> strains isolated from Mbuji-Mayi, lanes 45–48 = <i>T.b. gambiense</i> strains isolated from Masi-Manimba, lane 49 = <i>T.b. brucei</i> 427 WT, lane 50 = <i>T.b. brucei</i> 427 AT1/P2 KO, lane 51 = negative PCR control.</p

Restriction digest profile generated with AvaI PCR-RFLP on DNA of the <i>T.b. gambiense</i> strains as listed in Table 1, including the four strains isolated from relapsed mice.

<p>Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = <i>T.b. gambiense</i> strains isolated from Mbuji-Mayi, lane 45 = <i>T.b. gambiense</i> 15BT relapse 10 mg/kg BW, lane 46 = <i>T.b. gambiense</i> 163AT relapse 10 mg/kg BW, lane 47 = <i>T.b. gambiense</i> 346AT relapse 10 mg/kg BW, lane 48 = <i>T.b. gambiense</i> 346AT relapse 12 mg/kg BW, lane 49 = <i>T.b. gambiense</i> MBA, lane 50 = <i>T.b. gambiense</i> MM01, lane 51 = negative PCR control.</p

List of <i>T.b. gambiense</i> strains used in this study.

<p>In alias name: AT = after treatment, BT = before treatment. Treatment outcome: outcome of patient treated with melarsoprol (in Mbuji-Mayi) or with nifurtimox-eflornithine combination therapy (Masi-Manimba). Couple = number of the couple of two strains isolated from the same patient.</p><p>List of <i>T.b. gambiense</i> strains used in this study.</p

Amplicons generated with <i>AQP2/3</i>-PCR on DNA of the <i>T.b. gambiense</i> strains as listed in Table 1 and of the two <i>T.b. brucei</i> control strains.

<p>Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = <i>T.b. gambiense</i> strains isolated from Mbuji-Mayi, lanes 45–48 = <i>T.b. gambiense</i> strains isolated from Masi-Manimba, lane 49 = <i>T.b. brucei</i> 427 WT, lane 50 = <i>T.b. brucei</i> 427 AT1/P2 KO, lane 51 = negative PCR control.</p

Restriction digest profile generated with SduI PCR-RFLP on DNA of the <i>T.b. gambiense</i> strains as listed in Table 1, including the four strains isolated from relapsed mice.

<p>Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = <i>T.b. gambiense</i> strains isolated from Mbuji-Mayi, lane 45 = <i>T.b. gambiense</i> 15BT relapse 10 mg/kg BW, lane 46 = <i>T.b. gambiense</i> 163AT relapse 10 mg/kg BW, lane 47 = <i>T.b. gambiense</i> 346AT relapse 10 mg/kg BW, lane 48 = <i>T.b. gambiense</i> 346AT relapse 12 mg/kg BW, lane 49 = <i>T.b. gambiense</i> MBA, lane 50 = <i>T.b. gambiense</i> MM01, lane 51 = negative PCR control.</p