Clinical diversity and treatment results in Tegumentary Leishmaniasis: A European clinical report in 459 patients.

BACKGROUND: Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management. METHODOLOGY: Demographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients. PRINCIPAL FINDINGS: Compared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%). CONCLUSION/SIGNIFICANCE: CL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates.

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

Molecular identification of Leishmania spp. clinical isolates from Colombia based on hsp70 gene.

INTRODUCTION: Leishmaniasis is highly prevalent in Colombia, where at least six different species can cause disease of varying clinical presentations in humans. The identification of the infecting species is quite important for prognosis, therapeutics and epidemiology. Different techniques with variable discriminatory power have been used for the identification.  OBJECTIVE: To carry out the molecular identification of Leishmania species through the amplification of a fragment of the hsp70 gene.  MATERIALS AND METHODS: Molecular amplification of the hsp70 gene fragment (PCR-hsp70) followed by restriction fragment length polymorphism analysis (RFLP) was done for identification purposes using DNA from 81 clinical isolates of Leishmania.  RESULTS: A single amplicon was obtained for all samples analyzed. The enzymatic restrictions of the 81 PCR products identified 70 with a banding pattern corresponding to L. braziliensis with two different patterns (62 and eight isolates, respectively), nine isolates compatible with L. panamensis and two with L. guyanensis. The geographical origin of the isolates is consistent with previous reports about the distribution of the corresponding species in Colombia.  CONCLUSIONS: The PCR-hsp70/RFLP technique used is a valid tool for the identification of Leishmania species isolated from clinical samples of patients in Colombia, which may also be applicable to the study of strains obtained from vectors and reservoirs with epidemiological significance.

Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene.

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII+V+VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.

Detección de Leishmania spp. en base al gen que codifica la proteína HSP20 / Detection of Leishmania spp. based on the gene encoding HSP20

Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20) para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR).Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (ƙ=0,731), lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (Ƙ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.

Direct Leishmania species typing in Old World clinical samples: evaluation of 3 sensitive methods based on the heat-shock protein 70 gene.

In the diagnosis of leishmaniasis, identification of the causative Leishmania species is relevant for treatment, prognosis, and epidemiology. Three new hsp70-based PCR variants were developed and recently validated on clinical samples from Peru, without the need for culturing. We evaluated their performance on 133 clinical samples (bone marrow, blood, buffy coat, lymph node aspirates, lesion biopsies) from 42 cutaneous and 56 visceral leishmaniasis patients and 35 negative cases, all from Old World countries (Italy, Sudan, Israel, and Tunisia). The 3 new PCRs were significantly more sensitive than those previously described for hsp70, and their respective restriction fragment analyses were more efficient for species identification. In 79% of the parasitologically confirmed positive samples, the species could be identified directly from sample DNA. This evaluation demonstrated that these new tools are globally applicable in different geographical, clinical, and sampling contexts, and they could become the reference method for identification of Leishmania species in clinical specimens.

[Detection of Leishmania spp. based on the gene encoding HSP20]. / Detección de Leishmania spp. en base al gen que codifica la proteína HSP20.

OBJECTIVES: Explore a new target for molecular diagnosis of Leishmania. MATERIALS AND METHODS: We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. RESULTS: The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (κ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. CONCLUSIONS: The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.

Differentiation between Trypanosoma cruzi and Trypanosoma rangeli using heat-shock protein 70 polymorphisms.

OBJECTIVE: Differential diagnosis of infection with Trypanosoma cruzi or T. rangeli is relevant for epidemiological studies and clinical practice as both species infect humans, but only T. cruzi causes Chagas' disease. Their common antigen determinants complicate the distinction between both species, while current PCR assays used for differentiation show some drawbacks. We developed and validated a generic PCR discriminating the species by restriction fragment length polymorphism (RFLP) analysis and a duplex PCR specifically amplifying a differently sized fragment of both species. METHODS: The assays are based upon a partial region of the heat-shock protein 70 gene (hsp70). The analytical sensitivity and specificity were determined for both PCRs. The assays were analytically evaluated on a panel of six T. cruzi, one T. cruzi marinkellei and four T. rangeli strains, various other infectious pathogens, a panel of spiked samples of T. cruzi, and artificially mixed infections of T. cruzi and T. rangeli. Finally, the tools were applied on 36 additional isolates of Trypanosoma species. RESULTS: The detection limit of the PCRs was between 0.05 and 0.5 parasite genomes, and 1-10 parasites spiked in 200 µl blood. In artificial mixtures, PCR-RFLP picked up both species in ratios up to 10(2) and duplex PCR up to 10(4) . In the 36 isolates tested, both single and mixed infections were identified. All assays were shown to be specific. CONCLUSION: Our PCRs show high potential for the differential diagnosis of T. cruzi and T. rangeli, which in view of their sensitivity can aid in the confirmation of infection with these parasites in vectors, reservoirs and clinical samples.

HindII and SduI digests of heat-shock protein 70 PCR for Leishmania typing.

Restriction fragment length polymorphisms of the heat-shock protein 70 gene have been used for discriminating Leishmania species. Here, we validated HindII as a much cheaper alternative to EcoRII and SduI for discriminating Leishmania (Viannia) braziliensis from Leishmania (Viannia) naiffi and an atypical Leishmania (V.) braziliensis group, which was previously not possible.

Evolution and species discrimination according to the Leishmania heat-shock protein 20 gene.

The Leishmania genus comprises up to 35 species, of which 20 are responsible for human disease. However, the taxonomic status for many of them is under discussion. The small Heat Shock Proteins (sHSPs) are physiologically relevant, protecting cellular proteins from aggregation and maintaining cellular viability under intensive stress conditions. In Leishmania, a protein of this class was previously described, the 20-kDa heat-shock protein (HSP20), which is encoded by a single gene. In the present study, we used this target, alone or in combination with hsp70 gene, to investigate the phylogenetic relationships among Leishmania species. Using a pair of degenerate primers it was possible amplifying a 370bp fragment of the hsp20 coding region in 39 strains of very different geographic origins, representing in total 16 Leishmania species (14 if L. chagasi and L. archibaldi are considered synonymous names of L. infantum and L. donovani, respectively). Nucleotide sequences were readily obtained by direct sequencing of the amplification products. Both phylogenetic trees and networks based on either hsp20 sequences or combined datasets of hsp20 and hsp70 sequences were constructed. These phylogenic analyses supported the division of the Leishmania genus into nine species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis. Additionally, by network analysis, the subspecies L. (L.) donovani infantum and L. (V.) braziliensis peruviana were recognized within the L. (L.) donovani and L. (V.) braziliensis species, respectively. Therefore, hsp20 gene was found to be a suitable molecular marker for Leishmania typing and classification purposes. In addition, this study represents a solid contribution to the objective of establishing a more reliable taxonomy for the genus Leishmania.

Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters.

Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.

Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World.

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World.

Evolution of the Leishmania braziliensis species complex from amplified fragment length polymorphisms, and clinical implications.

In order to get more insight into its evolution and geographical distribution, we investigated the Leishmania (Viannia) braziliensis species complex using amplified fragment length polymorphisms and sequencing of a heat-shock protein 70 gene fragment. Previously, several assays had alluded to the high genetic diversity of the group, and single-locus assays typically identified two species, i.e. L. braziliensis and Leishmania peruviana, with occasional genetic signatures of both in the same strain. By analysis of 53 parasite isolates from Peru, and eight additional ones from other countries, we identified an atypical L. braziliensis cluster, and confirmed the origin of L. peruviana from the L. braziliensis cluster during the colonization of the western Andean coastal valleys. We discuss the clinical and taxonomical implications of our findings in relation to currently used species typing assays.

Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: assessment of a molecular stepwise approach.

We present an algorithm based on three PCR assays for Leishmania (Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.

Comparative gene expression analysis throughout the life cycle of Leishmania braziliensis: diversity of expression profiles among clinical isolates.

BACKGROUND: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study. METHODOLOGY/PRINCIPAL FINDINGS: We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered. CONCLUSION/SIGNIFICANCE: Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them.

Multilocus genotyping reveals high heterogeneity and strong local population structure of the Plasmodium vivax population in the Peruvian Amazon.

BACKGROUND: Peru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined. METHODS: Plasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (theta) were estimated. RESULTS: The proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IAS 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (theta = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies. CONCLUSION: The P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city.

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist.

Phylogeny of Leishmania species based on the heat-shock protein 70 gene.

The 70kDa heat-shock protein (HSP70) is conserved across prokaryotes and eukaryotes, and the protein as well as its encoding gene have been applied in phylogenetic studies of different parasites. In spite of the frequent use of New World Leishmania species identification on the basis of restriction fragment length polymorphisms (RFLP) in the hsp70 gene, it was never sequenced extensively for studying evolutionary relationships. To fill this void we determined the nucleotide sequence of an 1380bp fragment of the coding region commonly used in RFLP analysis, from 43 isolates and strains of different geographic origins. Combination with previously determined sequences amounted to a phylogenetic analysis including 52 hsp70 sequences representing 17 species commonly causing leishmaniasis both in the New and Old World. The genus Leishmania formed a monophyletic group with three distinct subgenera L. (Leishmania), L. (Viannia), and L. (Sauroleishmania). The obtained phylogeny supports the following eight species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis, in some of which subspecies can be recognized: L. (L.) donovani infantum, L. (V.) guyanensis panamensis, and L. (V.) braziliensis peruviana. The currently recognized L. (L.) aethiopica, L. (L.) garnhami, and L. (L.) amazonensis did not form monophyletic clusters. These findings are discussed in relation to results from other genes and proteins, which have to be integrated in order to build a genetically supported taxonomy for the entire genus.