Melarsoprol relapse rates and <i>TbAT1</i> genetic status, 1998 to present study.

a<p>Data from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000523#pntd.0000523-Barrett3" target="_blank">[13]</a>.</p>b<p>Use of melarsoprol discontinued in 2001.</p

PCR/RFLP patterns of selected samples from Moyo.

<p>U = undigested 677 bp fragment; W = wildtype pattern (STIB777S); M = Mutant (STIB777R); samples 1–11 are from patients M058, M052, M106, M048, M103, M074, M047, M044, M069, M062, and M031 respectively. Note the mutant pattern displayed in lanes 3, 5, 9, and the mixed genotype in lanes 6 and 10.</p

Regional Map of Uganda showing the origins (Districts/Counties) of study participants.

<p>Regional Map of Uganda showing the origins (Districts/Counties) of study participants.</p

Electrophoretic analysis of PCR-RFLP products.

<p>Representative Agarose gel results (A) <i>TbAT1</i> 677 bp PCR product of the trypanosome isolates from patients at Omugo (lanes B,D,E,G,I,J,K and N). The positive control (+) is a multidrug resistant clone STIB 950, negative control (−) was sterile water, while M is a 1 Kb molecular marker (Gene rulerTM, Fermentas, Life Sciences). (B) <i>Sfa NI</i> digest of the isolates from Omugo <i>TbAT1</i> 677 bp fragments. <i>TbAT1</i> wild type pattern of 566 bp and 111 bp fragments was displayed by all isolates from Omugo (lanes 1–10, 12–14), while the positive control (STIB 950) displayed the mutant genotype of 435 bp and 242 bp.</p

Growth of pyrimidine auxotrophic <i>T. b. brucei</i> bloodstream forms on various pyrimidine sources.

<p><i>PYR6-5</i>^−/− cell cultures were seeded at a density of 1×10⁵ cells ml⁻¹ and cultured at 37°C/5% CO₂, either in normal HMI-9 or in a simplified pyrimidine version supplemented with dialysed FBS and the indicated pyrimidine source at (A) 100 µM or (B) 1 mM. Samples were taken every 24 h and cell densities determined using a haemocytometer, in duplicate. The experiment shown is representative of several similar experiments with essentially identical results. In cultures with conditions that allowed fast growth, the trypanosome population declined after 36–48 h due to overgrowth.</p

Infectivity of pyrimidine auxotrophic <i>T. b. brucei</i>.

<p>(A) Survival of mice in groups of 6, each inoculated with 10⁵ bloodstream form trypanosomes of various clonal lines. (B) Parasitaemia of the same mice as depicted for survival in panel A. The average parasitaemia of the surviving mice is shown. Detection was by phase-contrast microscopy and detection limit was 1×10⁴; where the infected was sub-patent, a value of 5000 cells ml⁻¹ was inserted in order to arrive at a reasonable average. Both panels: ○, WT s427; ▪, <i>PYR6-5</i>^+/−; ▴, <i>PYR6-5</i>^−/−.</p

EC₅₀ values for some pyrimidine analogues tested on WT s427 and <i>PYR6-5</i>^−/− bloodstream forms grown in standard HMI-9, using a standard protocol based on the fluorescent indicator dye Alamar Blue.

<p>RF, resistance factor, being the ratio of the EC₅₀ values (µM) for knockout over WT strains. P value is based on an unpaired Students t-test.</p

Flow cytometry for DNA content in bloodstream form <i>PYR6-5</i>^−/− cells.

<p>Pyrimidine auxotrophic trypanosomes were either incubated in standard HMI-9 or, in parallel, in pyrimidine-free HMI-9^-tmd for up to 48 h, stained with propidium iodide and prepared for flow cytometric analysis. Whereas control cultures show a classical distribution of cells in G1, S and G2 phase, as well as a small percentage of cells with less than the normal diploid DNA content (debris, d), cells grown in pyrimidine-free medium showed a much higher percentage of cells with partial DNA content, and this increased dramatically between 24 and 48 h.</p

Quantitative analysis of DNA content in pyrimidine-starved trypanosomes.

<p>The peak area of G1, G2 and debris of flow cytometric analysis of DNA content (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058034#pone-0058034-g006" target="_blank">Fig. 6</a>) was determined using the ModFit software package after 24 or 36 of growth under various culturing conditions. Growth was in HMI-9 (control) or in HMI-9^-tmd with or without the addition of 100 µM of one pyrimidine as indicated. The data are the average of 3–6 independent determinations and statistical analysis was performed using one-way ANOVA with Tukey's correction (Prism 5, GraphPad). *, P<0.05; **, P<0.01.</p

Comparative expression of uridine phosphorylase in wild-type and pyrimidine auxotrophic trypanosomes.

<p>Expression of uridine phosphorylase (UP) was assessed by Real Time PCR in WT and <i>PYR6-5</i>^−/− strains grown under various conditions. The results are presented normalized to the control (group1) and are the average and SE of 8 replicates. 1. Control: WT grown in HMI-9; 2. WT grown 48 h in HMI-9^-tmd+100 µM uracil; 3. WT grown 48 h in HMI-9^-tmd+1 mM uridine; 4. PYR6-5^−/− grown 48 h in HMI-9^-tmd+100 µM uracil; 5. PYR6-5^−/− grown 48 h in HMI-9^-tmd+1 mM uridine; 6. PYR6-5^−/−long-term adapted to growth on uridine, grown 48 h in HMI-9^-tmd+100 µM uracil; 7. PYR6-5^−/−long-term adapted to growth on uridine, grown 48 h in HMI-9^-tmd+1 mM uridine. Data were analysed with a one-way ANOVA with Tukey's correction. Horizontal asterisks indicate significant differences from control; vertical asterisks indicate significant differences between individual bars as indicated.</p