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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.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2013 John Wiley & Sons Ltd.)

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., (© 2013.)

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., (Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

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.