Molecular Detection of Leishmania (Leishmania) mexicana in Sandflies from the State of Yucatan, Mexico.

Background: Leishmaniases are a group of vector-borne zoonotic diseases of public health relevance within the tropical and subtropical regions of the world. The state of Yucatan is a vulnerable and receptive area to localized cutaneous leishmaniasis (LCL) due to its proximity to the high-transmission endemic states of Campeche and Quintana Roo. Autochthonous cases of LCL caused by Leishmania (Leishmania) mexicana have been documented in the state, showing a geographical expansion of the disease. Materials and Methods: Using CO2-supplemented Centers for Disease Control and Prevention light traps and Shannon traps, we captured anthropophilic sandflies in the surroundings of a locality with recent records of autochthonous cases of LCL. Sandflies carrying Leishmania DNA were evidenced using PCR. Results: A total of 140 Phlebotominae (Diptera: Psychodidae) females of four species were captured: Lutzomyia (Tricholateralis) cruciata (Coquillett), Psathyromyia (Psathyromyia) shannoni (Dyar), Lutzomyia (Lutzomyia) longipalpis (Lutz and Neiva), and Dampfomyia (Coromyia) deleoni (Fairchild and Hertig). Molecular results showed that 6.1% (95% confidence interval [CI] = 2.3-12.9%) of Lu. cruciata and 43.8% (95% CI = 19.8-70.1%) of Pa. shannoni showed evidence of carrying L. (L.) mexicana DNA. Conclusion: We provide evidence of anthropophilic sandflies carrying L. mexicana DNA in a municipality with recorded autochthonous cases of LCL caused by this parasite species in the state of Yucatan, suggesting the emergence of new focus of LCL in Mexico.

Two phylogenetically divergent isocitrate dehydrogenases are encoded in Leishmania parasites. Molecular and functional characterization of Leishmania mexicana isoenzymes with specificity towards NAD+ and NADP.

Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD+, while Lmex_IDH-90 solely used NADP+ as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent Km value towards isocitrate (2.0 µM vs 43 µM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD+ and NADP+, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.

Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

Molecular diagnosis of Leishmania mexicana in a cutaneous leishmaniasis case in Sinaloa, Mexico.

Leishmaniasis has been considered endemic in Sinaloa, Mexico, since 1994. Despite that Leishmania mexicana is the main etiological agent of cutaneous leishmaniasis (CL) in other regions of Mexico, the species causing CL in patients from Sinaloa state has not been previously established, although Leishmania braziliensis has been found in the neighboring southern state, Nayarit. L. braziliensis is also associated with mucocutaneous leishmaniasis, which is a more complicated clinical variant. Due to the implications on individual and public health, the objective of this report was to identify the Leishmania species present in Sinaloa, Mexico. Using the first internal transcribed spacer (ITS-1) polymerase chain reaction-restriction fragment length polymorphism, we identified L. mexicana in a CL patient from Sinaloa and confirmed the extended distribution of this parasite in Mexico.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

Epidemiology of cutaneous leishmaniasis in Suriname: a study performed in 2006.

Cutaneous leishmaniasis (CL) is a widespread disease in Suriname caused by Leishmania Viannia guyanensis. It is argued that other Leishmania species are also responsible for CL and that the incidence is increasing. This study aimed to identify the species causing the disease and to estimate the annual detection rate of CL in Suriname in 2006. In Paramaribo, 152 patients were registered, of whom 33 were tested in two polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. Twenty-seven patients were infected with L. (V.) guyanensis (complex), one with L. (V.) lainsoni, and one with L. (Leishmania) amazonensis. In the hinterland, 162 CL suspected patients were registered by questionnaires; of these, 24 of 27 tested positive by PCR-RFLP (88.9%; 95% CI, 77.1-100%). With extrapolation of collected data, a detection rate was calculated of 5.32 to 6.13 CL patients per 1,000 inhabitants for the hinterland and 0.64 to 0.74 patients per 1,000 inhabitants for the whole country.

Leukotrienes are essential for the control of Leishmania amazonensis infection and contribute to strain variation in susceptibility.

Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa.

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.

Molecular probes and the polymerase chain reaction for detection and typing of Leishmania species in Mexico.

Leishmaniasis in Mexico is a public health problem because all the clinical forms have been recorded in most Mexican states. We studied patients showing clinical symptoms of any form of leishmaniasis, from several endemic areas. Bone marrow samples, aspirates or skin biopsies were taken and deoxyribonucleic acid (DNA) was extracted and amplified by the polymerase chain reaction (PCR) with universal primers AJS1 and DeB8, specific for the Leishmania subgenus Leishmania. The PCR products were then hybridized by dot- or Southern blotting and probed with probe 9.2, specific for the L. mexicana complex. If hybridization did not occur, the DNA was amplified with primers D1 and D2, specific for members of the L. donovani complex, and PCR products were hybridized with probe B4Rsa, also specific for the L. donovani complex. DNA was also amplified with primers B1 and B2, specific for the subgenus Viannia, and the PCR products were hybridized with probe B18, specific for the L. braziliensis complex. It was found that in Tabasco and Veracruz, Mexico, localized cutaneous leishmaniasis (LCL) is caused by infection with members of the L. mexicana complex, whereas in the states of Nayarit and Campeche it was due to infection with the L. mexicana and/or L. braziliensis complexes. Visceral leishmaniasis was caused by L. (L.) chagasi, mainly in the states of Chiapas and Guerrero, and by L. (L.) mexicana in one immunocompromised patient from Tabasco.

Use of DNA-based diagnostic methods for human leishmaniasis in Minas Gerais, Brazil.

DNA hybridisation was used to type 26 samples from lesions of human patients from the Rio Doce Valley (Minas Gerais, Brazil) clinically diagnosed as having cutaneous leishmaniasis, using kinetoplast DNA (kDNA) cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes. All samples were found to belong to the L. braziliensis complex. When biopsies were pressed directly onto touch blot membranes 38.5% of the samples were positive. The positivity and specificity obtained were both 100% when cultured blotted parasites were used. The results were confirmed by polymerase chain reaction (PCR) analysis using primers specific for the L. mexicana and L. braziliensis complexes.

Leishmaniose tegumental americana com extenso acometimento de mucosas: relato de caso e revisäo da literatura / American diffuse leishmaniosis with extensive attacking of mucous membranes: case report and literature review

A leishmaniose tegumentar americana é uma doença de evolução crônica que acomete, isoladamente ou em associação, a pele e as mucosas do nariz, boca, faringe e laringe (4). É causada por protozoários do gênero Leishmania e transmitida por insetos do gênero flebotomíneos. Os autores fazem uma revisão bibliográfica e relatam um caso de leishmaniose mucocutânea em um paciente de 26 anos, que apresentava lesão no palato duro e toda orofaringe, evoluindo com emagrecimento importante. A lesão foi diagnosticada através de biópsia de mucosa, a qual evidenciou o protozoário. O interesse do relato se dá pela ascensão do número de casos de leishmaniose em nosso meio, sendo importante o diagnóstico diferencial

Leishmania mexicana mexicana: genetic heterogeneity of mexican isolates revealed by restriction length polymorphism analysis of kinetoplast DNA.

Leishmania mexicana mexicana isolates from 23 patients with localized, diffuse, and an atypical "pseudodiffuse" form of cutaneous leishmaniasis were obtained in various endemic regions of Mexico. Restriction fragment length polymorphism analysis of kinetoplast DNA was done with nine different endonucleases in addition to an in vitro growth pattern analysis. We found that the 23 L. mexicana mexicana isolates could be consistently classified into six groups, according to the endonuclease digestion patterns obtained with HaeIII, HpaII, and MseI. Whereas localized cutaneous leishmaniasis isolates could have any of five patterns, diffuse cutaneous leishmaniasis showed only two patterns and pseudodiffuse cutaneous leishmaniasis consistently showed only one pattern. Thus, a clear correlation among digestion pattern, clinical disease, and geographical localization was obtained for the pseudodiffuse cutaneous leishmaniasis group. Additionally, the L. mexicana mexicana isolates could be differentiated into fast- and slow-growing groups. Diffuse cutaneous leishmaniasis isolates were found to be fast growing, whereas localized cutaneous leishmaniasis isolates fell into both categories. In contrast, all pseudo diffuse cutaneous leishmaniasis isolates were slow growing. Here we report the first study in which distinct and persistent genotypic characteristics of kinetoplast DNA heterogeneity within the L. mexicana mexicana species could be directly correlated with clinical disease and its growth behavior, suggesting that a distinctive restriction pattern could have important biological implications. Additionally, this study sheds new light on the biological significance of parasite kinetoplast DNA, since the heterogeneity seems not to be random but to form a distinct pattern.

Lectin-binding properties of different Leishmania species.

Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 x 10(6) receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 x 10(6) M(-1).

A new focus of cutaneous leishmaniasis due to Leishmania amazonensis in a Sub Andean region of Bolivia.

We detected a new outbreak focus with high incidence of cutaneous leishmaniasis in the Sub Andean region of La Paz. This area was never considered previously as an endemic zone of leishmaniasis. Leishmania stocks from human lesions were isolated: three stocks were explored by pulse field gradient electrophoresis, showing evidence for their affiliation to the L. mexicana complex. Eight stocks were submitted to isoenzyme electrophoresis and compared with five reference strains: L. amazonensis, L. braziliensis, L. chagasi, L. mexicana and L. pifanoi. Close genetic proximity was evidenced between newly isolated parasites and the reference stock of L. amazonensis, whereas high divergence was observed between them and either the L. pifanoi, L. mexicana, L. braziliensis and L. chagasi reference strains.

Identification of Mexican Leishmania species by analysis of PCR amplified DNA.

Leishmania parasites isolated into culture from patients with LCL or DCL from four different Mexican states were characterised using polymerase chain reaction (PCR), hybridisation with specific probes, and isoenzymes. PCR of the parasites showed that 10 of 11 of those isolates were members of the mexicana complex. This was confirmed in seven cases by isoenzymes. Restriction enzyme digests of PCR products of Mexican isolates showed the isolates to be different from the L.(L.) mexicana reference strain BEL21. Two (C2 and AM) of the isolates were shown to be a possible mixed infection between mexicana and braziliensis complex members. With a second set of samples from different patients from Campeche state, PCR of 14 biopsies indicated the presence of braziliensis complex members in six of the samples. The results showed that most of our isolates of Leishmania which come from the states of Tabasco and Veracruz are members of the Leishmania mexicana complex, but they seem to be different from the L.(L.) mexicana BEL21 reference strain. By hybridisation most of the biopsies (seven out of 14) from Campeche belong to the L. braziliensis complex and two out of 14 to L. mexicana complex and three out of 14 hybridised with both complexes, and two biopsies were negative. In Campeche, which is very close to Tabasco state and has border with Guatemala, we found members of the L. mexicana and L. braziliensis complexes.

Isoenzyme characterization of Leishmania isolated from human cases with localized cutaneous leishmaniasis from the State of Campeche, Yucatan Peninsula, Mexico.

Seventy-five isolates from the State of Campeche, Mexico, an area endemic for localized cutaneous leishmaniasis (LCL), were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as Leishmania (Leishmania) mexicana and 5 (6.7%) as L. (Viannia) braziliensis. This is the first report of authochthonus human LCL caused by L. (V.) braziliensis in the State of Campeche, Yucatan Peninsula, Mexico.

Coexistence of two species of Leishmania in the digestive tract of the vector Lutzomyia ovallesi.

Leishmania isolated from the digestive tract of a naturally infected Lutzomyia ovallesi sand fly were cultured in blood agar for rapid growth, cloning, and subsequent identification through schizodeme analysis, dot-blot hybridization, use of monoclonal antibodies with various specificities and absorbed polyclonal antibodies. Twenty-three clones isolated from the primary culture were identified. The results showed that parasites belonging to some clones corresponded to the L. mexicana complex, while others belonged to the L. braziliensis complex. These results clearly establish the coexistence of two Leishmania species in the digestive tract of a single Lu. ovallesi sand fly.

Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization.

The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy smears. By hybridizing the PCR product to new kDNA probes specific for either Leishmania mexicana or Leishmania braziliensis, we found that both species are major causes of cutaneous leishmaniasis in Venezuela, and the species identification was confirmed by restriction enzyme analysis of kDNA from biopsy cultures. This work demonstrates that PCR coupled with hybridization is useful not only for the diagnosis of cutaneous leishmaniasis but also for the taxonomic discrimination essential for both epidemiology and therapy. This technique can be used to diagnose leishmaniasis in a country in which the disease is endemic and can perhaps be adapted for use in a rural clinic.

Molecular karyotype characterization of Leishmania panamensis, Leishmania mexicana, and Leishmania major-like parasites: agents of cutaneous leishmaniasis in Ecuador.

Molecular karyotypes of Leishmania isolates from patients with cutaneous leishmaniasis in Ecuador were analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization. The DNA karyotypes of L. major-like parasites were similar between two human isolates from a lowland coastal and a highland Andean region, but were apparently different from those of eleven World Health Organization reference strains including L. major. The smallest chromosome of 240 kilobases in L. major-like parasites was found to belong to the 715-class of small linear chromosomal DNAs, which have been shown to appear in some lines of Leishmania. Chromosome banding patterns of L. mexicana isolates exhibited a novel, ordered, chromosomal ladder, and were identical among four human isolates and one canine isolate from a restricted geographic region in the Andes. On the other hand, minor chromosome size polymorphisms were observed among three L. panamensis isolates from different endemic regions near the Pacific Coast. Chromosomal locations of dihydrofolate reductase-thymidylate synthetase and P-glycoprotein genes revealed further differences in chromosomal organizations among these Leishmania species in Ecuador. These results indicate that karyotype analysis by PFGE is useful for epidemiologic studies of leishmaniasis in Ecuador.

Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in two cell compartments of Leishmania mexicana promastigotes. These activities could be attributed to two different isoenzymes, one residing in glycosomes, the other in the cytosol. We have cloned and sequenced the genes for both isoenzymes. The glycosomal enzyme is encoded by two tandemly linked genes of identical sequence and contains features frequently found in glycosomal enzymes: the presence of peptide insertions, a small carboxy-terminal extension with a potential glycosomal targeting signal (-SKM) and an excess of positively charged residues (net charge +7). Only one open reading frame was detected for the cytosolic enzyme. The amino acid sequences of the two proteins are only 55% identical. We discuss some evolutionary aspects of the observed organization of the GAPDH genes in the Trypanosomatidae and the role of the two isoenzymes in the metabolism of these organisms. The possibility to develop GAPDH-specific inhibitors that will be effective against the enzyme of various parasitic members of this family is explored.