S. De Doncker

Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World

INTRODUCTION Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. METHODS Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. RESULTS Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. CONCLUSION Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics

We describe Leishmania species determination on clinical samples on the basis of partial sequencing of the heat-shock protein 70 gene (hsp70), without the need for parasite isolation. The method is especially suited for use in non-endemic infectious disease clinics dealing with relatively few cases on an annual basis, for which no fast high throughput diagnostic tests are needed. We show that the results obtained from this gene are in nearly perfect agreement with those from multilocus enzyme electrophoresis, which is still considered by many clinicians and the World Health Organization (WHO) as the gold standard in Leishmania species typing. Currently, 203 sequences are available that cover the entire hsp70 gene region analysed here, originating from a total of 41 leishmaniasis endemic countries, and representing 15 species and sub-species causing human disease. We also provide a detailed laboratory protocol that includes a step-by-step procedure of the typing methodology, to facilitate implementation in diagnostic laboratories.

Plasticity of gp63 gene organization in Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana.

The genomic organization of gp63 genes in 4 and 7 isolates of Leishmania braziliensis and L. peruviana, respectively was studied by RFLP analysis with 3 restriction enzymes (Bgl I, Sal I and Apa I). Our results showed a marked polymorphism among isolates. Some characters were specific to L. braziliensis or to L. peruviana, and others specific to the respective biogeographical populations of L. peruviana. The average minimum copy number of gp63 genes was found to be higher in L. braziliensis (71) than in L. peruviana (46), suggesting that deletion of gp63 genes might be partially involved in the size decrease of the chromosome bearing gp63 genes, observed between those 2 species (from 700 to 610 kb). Our results may suggest the existence of at least 2 arrays of heterologous gp63 repeats, varying in relative copy number between L. braziliensis and L. peruviana, and among isolates of the latter species. Rearrangement of the gp63 genes was observed during long-term in vitro maintenance of a reference strain of L. braziliensis. These observations document the existence of a dynamic gp63 gene organization in Leishmania of the braziliensis complex.

Increased metacyclogenesis of antimony-resistant Leishmania donovani clinical lines

Mathematical models predict that the future of epidemics of drug-resistant pathogens depends in part on the competitive fitness of drug-resistant strains. Considering metacyclogenesis (differentiation process essential for infectivity) as a major contributor to the fitness of Leishmania donovani, we tested its relationship with pentavalent antimony (SbV) resistance in clinical lines. Different methods for the assessment of metacyclogenesis were cross-validated: gene expression profiling (META1 and SHERP), morphometry (microscopy and FACS), in vitro infectivity to macrophages and resistance to complement lysis. This was done on a model constituted by 2 pairs of reference strains cloned from a SbV-resistant and -sensitive isolate. We selected the most adequate parameter and extended the analysis of metacyclogenesis diversity to a sample of 20 clinical lines with different in vitro susceptibility to the drug. The capacity of metacyclogenesis, as measured by the complement lysis test, was shown to be significantly higher in SbV-resistant clinical lines of L. donovani than in SbV-sensitive lines. Together with other lines of evidence, it is concluded that L. donovani constitutes a unique example and model of drug-resistant pathogens with traits of increased fitness. These findings raise a fundamental question about the potential risks of selecting more virulent pathogens through massive chemotherapeutic interventions.

Karyotype plasticity in Neotropical Leishmania : an index for measuring genomic distance among L. (V.) peruviana and L. (V.) braziliensis populations

A method for phenetic analysis of karyotype data has been developed for Leishmania populations. Measurement of size difference between chromosomes recognized by a given DNA probe in different isolates led to the formulation of a Chromosome Size Difference Index (CSDI). The method was applied to phenetic analysis of 4 sets of chromosomes--each set being recognized by a different probe--in 37 L. (Viannia) peruviana isolates sampled along a North-South transect through the Peruvian Andes and, in 11 L. (V.) braziliensis isolates from the Amazonian forest (Peru, Bolivia and Brazil). Karyotype variability was better accounted for by CSDI than by a method based on disjunctive encoding of karyotype data. CSDI evidenced the nature of relationships between L. braziliensis and L. peruviana and it provided a coherent picture of geographical and genomic differentiation among parasite populations. The latter did cluster according to their geographical origin. L. braziliensis was found karyotypically more homogeneous than L. peruviana. Within L. peruviana, Northern populations were closer to L. braziliensis than to Southern L. peruviana populations. The validity of karyotypic populations, or karyodemes, was sustained.

Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony

INTRODUCTION Evaluation of Leishmania drug susceptibility depends on in vitro Sb(V) susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of Sb(V)-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as Sb(V)-resistant and -sensitive, in order to identify potential resistance markers. METHODS The differential expression of 13 genes involved in Sb(V) metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. RESULTS Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of Sb(V)-resistant compared to the group of Sb(V)-sensitive parasites (P<0.01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. DISCUSSION Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro Sb(V) resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro Sb(V) susceptibility assays, but interfering with the gene expression patterns.

Development and evaluation of different PCR-based typing methods for discrimination of Leishmania donovani isolates from Nepal.

INTRODUCTION Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. METHODS Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. RESULTS Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. CONCLUSION We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

Evolutionary history of Trypanosoma cruzi according to antigen genes

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.

Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia.

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.

In vitro promastigote fitness of putative Leishmania (Viannia) braziliensis/Leishmania (Viannia) peruviana hybrids.

In order to initiate studies on the phenotypic properties of hybrids vs. their putative parents, the in vitro growth behaviour of promastigotes was compared for 15 stocks characterised as Leishmania (Viannia) braziliensis, Leishmania (Viannia) peruviana and putative hybrids (isolated from the Eastern Andean valley of Huanuco, Peru). Five sets of three stocks, each set including a L.(V.)braziliensis, a L.(V.)peruviana and a putative hybrid, were constituted randomly and counted daily close to isolation from man (ten to 18 subcultures). Hybrids and L.(V.)peruviana presented similar growth characteristics, and they displayed a growth capacity (growth rate and cell density at stationary phase) significantly lower than the one of L.(V.)braziliensis. Following prolonged in vitro maintenance of one of the sets, the hybrid kept its lower growth capacity. The contrast between the difficulty to grow in vitro these putative hybrids, and their high isolation rate from natural populations is discussed.