Search

The PCR-ELISA represents a promising advance for diagnosis of visceral leishmaniasis (VL) in blood samples. However, the method has been validated mostly with HIV-positive patients who are known to have high levels of parasitaemia. We developed a new PCR-ELISA assay for specific detection of Leishmania in patients' blood and validated it in Nepalese subjects with clinically suspected VL, almost all of whom were HIV-negative. For blood samples, PCR-ELISA was more sensitive (83.9%) than conventional PCR (73.2%), and demonstrated 100% and 87.2% specificity when using healthy controls who had never travelled to a VL-endemic area and controls from a VL-endemic area as references, respectively. We have demonstrated the ability of PCR-ELISA to detect parasites in blood of HIV-negative patients. The method could be used for epidemiological as well as clinical purposes, as it reduces the need for traumatic bone marrow sampling and risky spleen aspiration.

Accurate identification of Leishmania species is important for monitoring clinical outcome, adequately targeting treatment, and evaluation of epidemiological risk in tegumentary leishmaniasis. This is especially the case in regions where several species coexist and for travel medicine where the geographical source of infection is not always obvious. Species identification presently depends on parasite isolation, which is not very sensitive and not necessarily representative of parasites actually present in human tissues. We evaluated a polymerase chain reaction (PCR) assay combining amplification of the gp63 genes and restriction fragment length polymorphism (RFLP) analysis (gp63 PCR-RFLP) for direct Leishmania species-identification in tissues collected from Peruvian patients in 1999. By comparison with a kinetoplast DNA-based PCR, our PCR assay showed a detection sensitivity of 85%. Three species were encountered among patient samples, Leishmania (Viannia) braziliensis, L. (V.) peruviana and L. (V.) guyanensis, and their frequency and geographical distribution corresponded to earlier epidemiological studies of leishmaniasis in Peru. However, unexpected results raised questions about (i) the contribution of human migration to the emergence of new foci of given species, (ii) the pathogenicity of some species, and (iii) the frequency of mixed or hybrid infections.

ATP-dependent protease complexes are present in all three kingdoms of life, where they rid the cell of misfolded or damaged proteins and control the level of certain regulatory proteins. They include the proteasome in Eukaryotes, Archea, and Actinomycetales and the HslVU (ClpQY) complex in other eubacteria. We showed that genes homologous to eubacterial HslV (ClpQ) and HslU (ClpY) are present in the genome of trypanosomatid protozoa and are expressed. The features of the cDNAs indicated that bona fide trypanosomatid messengers had been cloned and ruled out bacterial contamination as the source of the material. The N-terminal microsequence of HslV from Leishmania infantum (Protozoa: Kinetoplastida) permitted the identification of the propeptide cleavage site and indicated that an active protease is present. High similarities (> or =57.5%) with the prototypical HslV and HslU from Escherichia coli and conservation of residues essential for biochemical activity suggested that a functional HslVU complex is present in trypanosomatid protozoa. The structure of the N-termini of HslV and HslU further suggested mitochondrial localization. Phylogenetic analysis indicated that HslV and HslU from trypanosomatids clustered with eubacterial homologs but did not point to any particular bacterial lineage. Because typical eukaryotic 20S proteasomes are present in trypanosomatids, we concluded that the eubacterial HslVU and the eukaryotic multicatalytic protease are simultaneously present in these organisms. To our knowledge this is the first report of a eubacterial HslVU complex in eukaryotes and, consequently, of the simultaneous occurrence of both a proteasome and HslVU in living cells.

This paper reviews our exploration of the dynamics of the Leishmania genome and its contribution to epidemiology and diagnosis. We used as a model Peruvian populations of L. (Viannia) braziliensis and L. (V.) peruviana, 2 species very close phylogenetically, but phenotypically very different in biotope and pathology. We initially focused on karyotype analysis. Our data showed that chromosomes were subject to a fast rate of evolution, and were sensitive indicators of genetic drift. Therefore, molecular karyotyping appeared an adequate tool for monitoring (i) emergence of close species, (ii) ecogeographical differentiation at the intraspecific level, and (iii) strain 'fingerprinting'. Chromosome size variation was mostly due to the number of tandemly repeated genes (rDNA, mini-exon, gp63, and cysteine proteinase genes), and could involve the deletion of unique genes (L. (V.) braziliensis-specific gp63 families). Considering the importance of these genes in parasitism, their rearrangement might have functional implications: adaptation to different environments and pleomorphic pathogenicity. Our knowledge of genome structure and dynamics was used to develop new polymerase chain reaction (PCR) techniques. Amplification of gp63 genes followed by cleavage with restriction enzymes and study of restriction fragment length polymorphism (gp63 PCR-RFLP) allowed the discrimination of all species tested, even directly in biopsies with 95% sensitivity (compared with PCR amplification of kinetoplast deoxyribonucleic acid). At the intra-specific level, RFLP was also observed and corresponded to mutations in major immunogen domains of gp63. These seem to be under strong selection pressure, and the technique should facilitate addressing how the host's immune pressure may modulate parasite population structure. Altogether, gp63 PCR-RFLP represents a significant operational improvement over the other techniques for molecular epidemiology and diagnosis: it combines sensitivity, discriminatory power and prognostic value.

Visceral leishmaniasis (VL), also known as kala-azar, is a vector-borne disease caused by a protozoan of the Leishmania donovani complex. A phlebotomine sandfly transmits the parasite from person to person or via an animal reservoir. VL is a severe, debilitating disease, characterized by prolonged fever, splenomegaly, hypergammaglobulinaemia and pancytopenia. Patients become gradually ill over a period of a few months, and nearly always die if untreated. Case-fatality ratios are high even in treated patients. Worldwide an estimated 500,000 VL cases occur each year. This study reviews clinical, epidemiological and public health aspects of the disease and shows how critical adequate case detection is for the success of VL control. Examination of the issue of VL diagnosis with respect to the global challenges in VL control leads to the observation that a sound diagnostic-therapeutic algorithm for the health services in endemic areas is badly needed. Serological tests could be an alternative to parasitological diagnosis and the direct agglutination test (DAT) was found to fulfil many criteria for a 'field test', including cost effectiveness. Although research needs on vaccine and better drugs continue to be high on the agenda, a VL test-treatment strategy based on currently available highly sensitive serological tests, such as the DAT, should be introduced in the health services in endemic areas.

In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, is due to a stage-specific accumulation of the respective mRNAs. Through the targeting of a reporter construct in the procyclin promoter region, we show that independently of any selection pressure, a relatively high level of transcription (approximately 10%) occurs from the procyclin promoter in the bloodstream form. This transcription leads to the production of detectable amounts of polyadenylated mRNAs. However, RNA elongation in the procyclin transcription unit is down-regulated at this stage. Transcription elongation in the procyclin and VSG units is inversely controlled by the combination of factors which cause the differentiation of bloodstream into procyclic forms in vitro. These factors include temperature, citrate/cis-aconitate and the incubation medium. Our results suggest that inverse regulations of primary transcription in the VSG and procyclin units are early events that underly the differentiation of the parasite.

We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for kinetoplastid protozoa, the Sh ble, or phleomycin, resistance gene. A plasmid containing this gene targeted to the tubulin gene locus by homologous sequences was introduced into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance. Southern analysis of stable transformants showed that the plasmid had been integrated into the tubulin locus by homologous recombination. Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed in these forms in the absence of selective drug pressure. In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulin promoter, despite the presence of a VSG promoter in the integrated construct. The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.

A recently described simple kit for isolating African trypanosomes in vitro (KIVI) was tested further with blood samples from man and other animals in Côte d'Ivoire and République du Congo. A high rate of success was achieved, with positive cultures being found 5-36 d after inoculation. The method was also of value in diagnosis. Parasitaemia was initially detected by the haematocrit method; in addition, the mini-anion exchange column was used for human blood and lymph fluid from patients with swollen glands was examined. The card agglutination test (CATT) was applied to the human blood samples. In Côte d'Ivoire, all 5 parasitaemic patients, who were also positive by CATT, yielded positive KIVI cultures. Of 15 animals, 2 parasitaemic and 10 apparently aparasitaemic individuals gave positive cultures. In the Congo, none of the 22 animals was parasitaemic and none gave a positive culture. Of 647 human subjects initially screened, 61, mostly with a positive CATT, were examined by KIVI; 20 gave positive cultures. Seven of these cultures originated from patients in whom no trypanosome had been seen in blood or lymph fluid, although blood from 2 parasitaemic patients failed to yield positive KIVI cultures. Some patients with CATT-negative whole blood and/or serum were positive by KIVI.

The identification of antigen types in tsetse salivary gland metacyclic populations of Trypanosoma brucei requires the production of monospecific antisera to the corresponding bloodstream variable antigen types. Monospecific antisera against clones from cyclically transmitted populations are difficult to prepare, however, owing to the antigenic lability of such clones. This problem has been overcome by isolating an antigenically stable clone from a syringe-infected rabbit at a time when its serum showed incipient activity towards metacyclic trypanosomes. Monospecific antisera raised against this clone reacted with up to 20% metacyclics in trypanolysis and immunofluorescence tests, confirming that a clone-derived metacyclic population of T. brucei is heterogeneous with respect to variable antigen type.