Prospective evaluation of a rapid diagnostic test for Trypanosoma brucei gambiense infection developed using recombinant antigens.

BACKGROUND: Diagnosis and treatment are central elements of strategies to control Trypanosoma brucei gambiense human African trypanosomiasis (HAT). Serological screening is a key entry point in diagnostic algorithms. The Card Agglutination Test for Trypanosomiasis (CATT) has been the most widely used screening test for decades, despite a number of practical limitations that were partially addressed by the introduction of rapid diagnostic tests (RDTs). However, current RDTs are manufactured using native antigens, which are challenging to produce. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this study was to evaluate the accuracy of a new RDT developed using recombinant antigens (SD BIOLINE HAT 2.0), in comparison with an RDT produced using native antigens (SD BIOLINE HAT) and CATT. A total of 57,632 individuals were screened in the Democratic Republic of the Congo, either passively at 10 health centres, or actively by 5 mobile teams, and 260 HAT cases were confirmed by parasitology. The highest sensitivity was achieved with the SD BIOLINE HAT 2.0 (71.2%), followed by CATT (62.5%) and the SD BIOLINE HAT (59.0%). The most specific test was CATT (99.2%), while the specificity of the SD BIOLINE HAT and SD BIOLINE HAT 2.0 were 98.9% and 98.1%, respectively. Sensitivity of the tests was lower than previously reported, as they identified cases from partially overlapping sub-populations. All three tests were significantly more sensitive in passive than in active screening. Combining two or three tests resulted in a markedly increased sensitivity: When the SD BIOLINE HAT was combined with the SD BIOLINE HAT 2.0, sensitivity reached 98.4% in passive and 83.0% in active screening. CONCLUSIONS/SIGNIFICANCE: The recombinant antigen-based RDT was more sensitive than, and as specific as, the SD BIOLINE HAT. It was as sensitive as, but slightly less specific than CATT. While the practicality and cost-effectiveness of algorithms including several screening tests would need to be investigated, using two or more tests appears to enhance sensitivity of diagnostic algorithms, although some decrease in specificity is observed as well.

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase.

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).

Luminescent multiplex viability assay for Trypanosoma brucei gambiense.

BACKGROUND: New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient viability assay to monitor drug efficacy. METHODS: In this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex viability assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin viability assay for drug sensitivity testing of T.b. gambiense. RESULTS: In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 10(4) cells ml(-1) for the Renilla luciferase measurement and 5 × 10(3) cells ml(-1) for the ATP measurement. Both assays of the LMVA showed linearity up to 10(6) cells ml(-1) and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin viability assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both viability assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both assays of the LMVA. CONCLUSIONS: LMVA has some advantages for viability measurement of T.b. gambiense: it requires less incubation time for viability detection than the fluorimetric resazurin assay and in LMVA, two sensitive and independent viability assays are performed in the same experiment.

A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes.

The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH(4)Cl) then allows precise titration of steady state lysosomal pH (4.84+/-0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa.

Glycosylinositolphosphate soluble variant surface glycoprotein inhibits IFN-gamma-induced nitric oxide production via reduction in STAT1 phosphorylation in African trypanosomiasis.

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-gamma, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-gamma vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-gamma followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-alpha, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-gamma stimulation resulted in a marked reduction of IFN-gamma-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-gamma-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-gamma response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.

Repetitive proteins from the flagellar cytoskeleton of African trypanosomes are diagnostically useful antigens.

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection.

Distribution of filipin-sterol complexes in the bloodstream form of Trypanosoma brucei gambiense.

The polyene antibiotic, filipin, was used as a probe for demonstrating sterols in the plasma membrane of Trypanosoma brucei gambiense. Three different regions of the continuous plasma membrane over the cell body proper, flagellar pocket, and flagellum, were compared as to density and distribution of the filipin-sterol complexes by freeze-fracture method. The density of the complexes was highest in the cell body membrane and lowest over the flagellar pocket. The filipin-sterol complexes in the cell body membrane were distributed homogeneously on both the protoplasmic and exoplasmic faces except in the zone of flagellar attachment. This junctional zone showed a linear, complex-poor region. In the flagellar membranes, a line of complex-poor regions was observed along the juncture of the flagellum to the cell body. At the neck of the flagellar pocket, the membranes of the flagellar pocket and flagellum were closely opposed, with few filipin-sterol complexes on either membrane. At the base of the flagellar shaft, the complexes were completely lacking on both faces. Based on these observations, the zones of juncture observed in T.b. gambiense seems to be similar in ultrastructure to mammalian cell junctions, such as tight, gap, septate junctions and desmosomes, which show a nearly complete absence of the filipin-sterol complexes.