Philippe Truc

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers.

Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group-specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group I.

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo‐encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 °C. CSF end‐titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme‐linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut‐off value of ≥ 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end‐titres ≥ 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end‐titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine.

Distinct roles of haptoglobin-related protein and apolipoprotein L-I in trypanolysis by human serum

Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Trypanosoma brucei brucei by forming anion-selective pores in the lysosomal membrane of the parasite. Another HDL component, haptoglobin-related protein (Hpr), has been suggested as an additional toxin required for full trypanolytic activity of normal human serum. We recently reported the case of a human lacking apoL-I (apoL-I−/−HS) as the result of frameshift mutations in both apoL-I alleles. Here, we show that this serum, devoid of any trypanolytic activity, exhibits normal concentrations of HDL-bound Hpr. Conversely, the serum of individuals with normal HDL-bound apoL-I but who lack Hpr and haptoglobin [Hp(r)−/−HS] as the result of gene deletion (anhaptoglobinemia) exhibited phenotypically normal but delayed trypanolytic activity. The trypanolytic properties of Hp(r)−/−HS were mimicked by free recombinant apoL-I, whereas recombinant Hpr did not affect trypanosomes. The lysis delay observed with either Hp(r)−/−HS or recombinant apoL-I could entirely be attributed to a defect in the uptake of the lytic components. Thus, apoL-I is responsible for the trypanolytic activity of normal human serum, whereas Hpr allows fast uptake of the carrier HDL particles, presumably through their binding to an Hp/Hpr surface receptor of the parasite.

Atypical human infections by animal trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

First isolation of Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the tsetse fly (Glossina palpalis palpalis) midgut

This paper reports the first evidence of the presence of bacteria, other than the three previously described as symbionts, Wigglesworthia glossinidia, Wolbachia, and Sodalis glossinidius, in the midgut of Glossina palpalis palpalis, the tsetse fly, a vector of the chronic form of human African trypanosomiasis in sub-Saharan African countries. Based on the morphological, nutritional, physiological, and phylogenetic results, we identified Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the midgut of the tsetse fly from Angola. Enterobacter spp. was the most frequently isolated. The role of these bacteria in the gut, in terms of vector competence of the tsetse fly, is discussed, as is the possibility of using these bacteria to produce in situ trypanolytic molecules.

Trypanosoma brucei gambiense Type 1 populations from human patients are clonal and display geographical genetic differentiation.

We have rigorously tested the hypothesis that Trypanosoma brucei gambiense Type 1 is composed of genetically homogenous populations by examining the parasite population present in Human African Trypanosomiasis (HAT) patients from the Democratic Republic of Congo (DRC) and Cameroon (CAM). We amplified eight microsatellite markers by PCR directly from blood spots on FTA filters, thereby avoiding the significant parasite selection inherent in the traditional isolation techniques of rodent inoculation or in vitro culture. All microsatellite markers were polymorphic, although for four markers there was only polymorphism between the DRC and CAM populations, not within populations, suggesting very limited genetic exchange. Within the largest population from the DRC, Hardy-Weinberg equilibrium is not evident at any loci. This evidence suggests a clonal population. However, there was significant sub-structuring between the DRC and CAM samples (F(ST) = 0.32), indicating that Trypanosoma brucei gambiense Type 1 has genetically distinct clades. The data combine to indicate that genetic exchange plays a very limited role. The finding of distinct clades in different places suggests the possibility that samples from humans with clinical signs represent clonal expansions from an underlying population that requires identifying and characterising.

Multilocus isozyme identification of Trypanosoma brucei stocks isolated in Central Africa: evidence for an animal reservoir of sleeping sickness in Congo

Six Congolese and 3 Zairian Trypanosoma brucei stocks were studied by isozyme cellulose acetate electrophoresis. Twenty isozyme systems were used, of which only 5 showed variability. These 5 polymorphic systems made it possible to identify 5 different zymodemes. Zymodemes isolated from man were recorded both from pig and sheep too, which confirms the results of previous authors. This favors the existence of an animal reservoir of human African trypanosomiasis in the Congo, which could play a role in the transmission of the disease, at least by the maintenance of residual foci.

Genetic variability within Trypanosoma brucei gambiense: evidence for the circulation of different genotypes in human African trypanosomiasis patients in Côte d'Ivoire

For 23 Ivoirian patients infected by Trypanosoma-brucei gambiense, isolation and genetic characterization using PCR and microsatellite primers were performed (in 1996-99) using 2 different isolates (A and B) from each patient. When using TBDAC 1/2, 7 genotypes were observed, and DNAs A and B for 2 patients were different. This might be the first evidence of the presence of 2 different genotypes of T. b. gambiense group 1 in the same patient.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 μg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 μg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Human African trypanosomiasis in Angola: clinical observations, treatment, and use of PCR for stage determination of early stage of the disease

Biological and clinical observations are described for 224 patients infected by human African trypanosomiasis (HAT) in Angola in 2007 and 2008. Seven patients were initially classified in stage 1 (S1), 17 intermediate stage (IS) (WBC <20 lymphocytes/μl with absence of trypanosomes in cerebrospinal fluid (CSF) and no neurological signs), and 200 in stage 2 (S2). Out of 224 patients, 165 (73.6%) presented one or more neurological signs. During treatment with eflornithine, six deaths of S2 patients occurred, five of which were because of an encephalopathy syndrome. Nine patients were diagnosed with a relapse or suspected treatment failure during the follow-up: eight patients after treatment with eflornithine (relapse rate 4.1%) and one patient after pentamidine (6.6%). The contribution of PCR for stage determination evaluated for S1 and IS confirms the difficulty of stage determination, as one S1 patient and two IS patients were carriers of trypanosomes detected a posteriori by PCR in CSF but were treated with pentamidine while follow-up did not confirm treatment efficacy. Since 2001 in Angola, either by passive or active mode detection, approximately 80% of the new cases every year were in S2, whereas the annual number of cases has regressed, probably because the transmission of HAT is decreasing. However, stage determination and treatment remain two major issues for the chronic form of sleeping sickness.