Brice Rotureau

Use of PCR-Restriction Fragment Length Polymorphism Analysis To Identify the Main New World Leishmania Species and Analyze Their Taxonomic Properties and Polymorphism by Application of the Assay to Clinical Samples

ABSTRACT At least 13 characterized Leishmania species are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment length polymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW) Leishmania species from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevant Leishmania species with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NW Leishmania species typing rapidly and easily in the field constitutes a very valuable improvement for detection of Leishmania spp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.

Through the dark continent: African trypanosome development in the tsetse fly.

African trypanosomes are unicellular flagellated parasites causing trypanosomiases in Africa, a group of severe diseases also known as sleeping sickness in human and nagana in cattle. These parasites are almost exclusively transmitted by the bite of the tsetse fly. In this review, we describe and compare the three developmental programs of the main trypanosome species impacting human and animal health, with focus on the most recent observations. From here, some reflections are made on research issues concerning trypanosome developmental biology in the tsetse fly that are to be addressed in the future.

The flagellum-mitogen-activated protein kinase connection in Trypanosomatids: a key sensory role in parasite signalling and development?

Trypanosomatid parasites are the causative agents of severe human diseases such as sleeping sickness, Chagas disease and leishmaniases. These microorganisms are transmitted via different insect vectors and hence are confronted to changing environments during their infectious cycle in which they activate specific and complex patterns of differentiation. Several studies in Trypanosoma brucei and in different subspecies of Leishmania have shed light on the role of mitogen‐activated protein (MAP) kinases in these processes. Surprisingly, several MAP kinases turned out to be involved in the control of flagellum length in the promastigote stage of Leishmania. Recently, a sensory function has been recognized for cilia and flagella in unicellular and multicellular eukaryotes. This review aims to stimulate discussions on the possibility that the Trypanosomatid flagellum could act as a sensory organ through the MAP kinase pathway, with the objective to encourage investigation of this new hypothesis through a series of proposed experimental approaches.

Molecular Epidemiology of Leishmania (Viannia) guyanensis in French Guiana

ABSTRACT Little information is available about the genetic variability of Leishmania populations and the possible correlations with ecoepidemiological features of leishmaniases. The present study was carried out in French Guiana, a country where cutaneous leishmaniases (CL) are endemic over the whole territory. The genetic polymorphism of a nuclear sequence encompassing the end of the ribosomal small subunit and the internal transcribed spacer 1 of 265 isolates from patients with CL was examined by restriction fragment length polymorphism analysis. Genotypes based on the fingerprinting phenetic integration were compared to epidemiological, clinical, and geographical data. In agreement with previous reports, five different Leishmania species were identified, but Leishmania (Viannia) guyanensis represented 95.8% of the samples. Two distinct L. (V.) guyanensis populations were found to originate in two ecologically characterized regions. Higher lesional parasite densities and the need for additional treatments were significantly linked to genotype group I. Parasites of genotype group II were more likely to cause chronic and disseminated cutaneous forms in patients. L. (V.) guyanensis was previously said not to be very polymorphic; however, the present analysis resulted in a significant degree of discrimination among L. (V.) guyanensis isolates from diverse ecological areas and with different clinical implications.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology. DOI: http://dx.doi.org/10.7554/eLife.17716.001

Molecular bases of cytoskeleton plasticity during the Trypanosoma brucei parasite cycle

African trypanosomes are flagellated protozoan parasites responsible for sleeping sickness and transmitted by tsetse flies. The accomplishment of their parasite cycle requires adaptation to highly diverse environments. These transitions take place in a strictly defined order and are accompanied by spectacular morphological modifications in cell size, shape and positioning of organelles. To understand the molecular bases of these processes, parasites isolated from different tissues of the tsetse fly were analysed by immunofluorescence with markers for specific cytoskeleton components and by a new immunofluorescence‐based assay for evaluation of the cell volume. The data revealed striking differences between proliferative stages found in the midgut or in the salivary glands and the differentiating stage occurring in the proventriculus. Cell proliferation was characterized by a significant increase in cell volume, by a pronounced cell elongation marked by microtubule extension at the posterior end, and by the production of a new flagellum similar to the existing one. In contrast, the differentiating stage found in the proventriculus does not display any increase in cell volume neither in cell length, but is marked by a profound remodelling of the posterior part of the cytoskeleton and by changes in molecular composition and/or organization of the flagellum attachment zone.

Forward motility is essential for trypanosome infection in the tsetse fly

African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.

Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body

ABSTRACT The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

First Report of Leishmania infantum in French Guiana: Canine Visceral Leishmaniasis Imported from the Old World

ABSTRACT The first two cases of canine visceral leishmaniasis in French Guiana are described. One infected dog was most probably imported from France. A second dog was then infected with Leishmania infantum in French Guiana. These observations exemplify the intercontinental transportation theory for L. infantum.

Leishmaniasis among gold miners, French Guiana.

To the Editor: In 2004, the Cayenne General Hospital and public health centers recorded 348 new cases of cutaneous leishmaniasis (CL) in French Guiana (1). A case of CL was considered confirmed if cutaneous lesions were present for >2 weeks; the patient had a compatible epidemiologic history; and microscopic examination of dermal scrapings, parasite cultivation, or both showed positive results for Leishmania. According to the population estimate given by the French National Institute for Statistics and Economical Studies (INSEE, Cayenne), the incidence of CL in 2004 was 0.2%–0.4% and has been relatively stable since 1979 (2,3). However, when the annual number of cases per village were examined, new CL cases were heterogeneously distributed. Saint Elie, a gold-mining village in the inland neotropical forest, had an apparent incidence rate of 25.9% in 2004 and 28.9% in 2005 (Figure); risk for infection in this village was, on average, 65× higher than anywhere else in French Guiana. We tested samples from 12 random CL patients with a Leishmania-specific polymerase chain reaction–restriction fragment length polymorphism test that targeted the internal transcribed spacer 1 of ribosomal RNA genes with primers SSU-12103-D (5´-GGGAATATCCTCAGCACGT-3´) and 5.8S-13333-R (5´-CGACACTGAGAATATGGCATG-3´) (4). All these patients were infected with Leishmania guyanensis. Figure Number of new cutaneous leishmaniasis (CL) cases registered in health centers of 4 villages of French Guiana (Iracoubo, Regina, Saul, and Saint Elie) from 2000 to 2005. For each village, the 1999 population estimate (French National Institute ... Isolated in dense rainforest (no road or airport) and with 239 inhabitants (INSEE, Cayenne), Saint Elie is situated on a gold seam; miners illegally create trails from the village to deposits in a 10-km circumference in the dense forest around the village. Compared to other French Guianan villages, such as Saul and Regina, which are similarly isolated in the rainforest and have 160 and 765 inhabitants (INSEE, Cayenne), respectively, and Iracoubo, the village closest to Saint Elie with 1,430 inhabitants (INSEE, Cayenne), substantially more new CL cases have been observed in Saint Elie since 2003. Since 2000, medical rounds have been undertaken every 15 days in the villages of Saint Elie and Saul, whereas people from Regina and Iracoubo have doctors at their disposal every day. Official records indicate that the population of Saint Elie has doubled in the past 10 years, reaching 239 inhabitants in 1999 (INSEE, Cayenne). However, 860 new medical files have been registered in the Saint Elie Health Centre since 2000. This finding could be explained by the high number of illegal workers in this area. Patient interviews showed that most of these workers (≈90%) originated from the poorest northern Brazilian states (Para, Amapa, Roraima, and especially Maranhao). Thus, the incidence rate of 25.9%, calculated on the basis of 239 inhabitants, was likely overestimated. Taking into account a substantial turnover in migrant populations, the denominator could be 500–1,000 inhabitants, and the incidence rate would be 6.2%–12.4%. All patients worked in the small-scale gold mines surrounding Saint Elie, and CL cases were recorded without seasonal fluctuations. Imported cases are possible, but reports are likely to be anecdotal because clinical observations, estimated dates of infection, and duration of patient stay in Saint Elie were congruent and because all genotyped strains were Guianan L. guyanensis (1). Several infection risk factors exist simultaneously in this situation. In a CL-endemic area, immigrant populations, who are mostly nonimmune, exert pressure on the environment (deforestation) that directly increases their risk for exposure to infected vectors, in the absence of prophylactic measures. The initial short-term effect of deforestation is the mobilization of aggressive adult sandflies, which have been disturbed while resting. However, the ability of zoophilic vectors to adapt to peridomestic environments has also already greatly influenced the distribution of leishmaniases in South America (5–7). Considering the uncertainty of the population estimate, turnover, and immunity status, we assume that incidence rates should be considered cautiously. Nevertheless, we found that gold mining in forested areas constitutes a risk factor for CL, at least in French Guiana and probably in all Amazonian rainforests. This risk could be a public health concern. Larger studies in other gold-mining areas are required to quantify the incidence of CL among workers to effectively focus prophylactic and preventive campaigns.