Strains Of Leishmania Research Articles

Natural products derived steroids as potential anti-leishmanial agents; disease prevalence, underlying mechanisms and future perspectives.

Leishmaniasis is a vector-borne infection caused by protozoan parasites from the genus leishmania and is among the most neglected tropical diseases. It is highly prevalent disease, affecting about 350 million population worldwide. Only limited number of anti-leishmanial agents are approved for clinical use till now and they are associated with side effects and have limited efficacy. Subsequently, natural products based discovery of more safe and effective drugs against leishmania is under scientific consideration. Various studies reported the efficacy of natural products against intracellular and extracellular forms of leishmania species. This work is aimed to evaluate current literature focused on the anti-leihmanial efficacy of steroidal moieties from natural products and their mechanism of action. Compounds including steroidal saponins, steroidal alkaloids and phytosterols were found to exhibit considerable anti-leishmanial efficacy. For instance, steroidal saponin, (25R)-spirost-5-en-3b-ol,3-O-α-rhamnopyranosyl-(1→4)-α-rhamnopyranosyl-(1→4)-[a-rhamnopyranosyl-(1→2)]-glucopyranoside isolated from A. paradoxum has completely eradicated Leishmania major promastigotes at 50µgmL-1 dose. Spirostanic saponins isolated from Solanum paniculatum L. were effective against Leishmania amazonensis promastigotes. Turgidosterones isolated from Panicum turgidum exhibited high leishmanicidal potentials against Leishmania donovani promastigotes with IC50 of 4.95-8.03µgmL-1 and even better activity against amastigotes exhibiting an IC50 of 4.50-9.29µgmL-1. Likewise, racemoside-A from Asparagus racemosus was found effective against an antimonial sensitive (AG83) and antimonial resistant (GE1F8R) strains of the L. donovani. Moreover, steroidal alkaloids including hookerianamide-1, hookerianamide-H, hookerianamide-J, hookerianamide-K, dehydrosarsalignone, vagenine-A, sarcovagine-C, holaphylline, saracodine, holamine, 15-α hydroxyholamine, holacurtin, N-desmethyl holacurtine and elasticine has exhibited time and dose-dependent efficacy against various strains of leishmania. β-sitosterol was found active against multiple strains of leishmania. These compounds mainly exhibit their therapeutic efficacy via liberation of ROS, mitochondrial depolarization, morphological and ultra-structural changes, accumulation of lipid droplets, depletion of non-protein thiols and triggering apoptotic pathways. In conclusion, leishmaniasis is a major health problem in many countries. Plants-derived steroids moieties have reveled efficacy against leishmaniasis and is a source of lead compounds. Further detailed molecular studies are warranted for the discovery of more effective and safe anti-leishmanial drugs.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

Leishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies. Phagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79. Proteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species.

First Example of Antiparasitic Activity Influenced by Thermochromism: Leishmanicidal Evaluation of 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine Metal Complexes.

The World Health Organization catalogues illnesses such as Leishmaniasis as neglected diseases, due to low investment in new drugs to fight them. The search of novel and non-side effects anti-parasitic compounds is one of the urgent needs for the Third World. The use of triazolopyrimidines and their metallic complexes has demonstrated hopeful results in this field. This work studies the antiparasitic efficacy of a series of 5,7-dimethyl-1,2,4- triazolo[1,5-a]pyrimidine first row transition metal complexes against three leishmania spp. strains. The in vitro antiproliferation of promastigote forms of different strains of leishmania spp. (L. infantum, L. braziliensis and L donovani) and the cytotoxicity in macrophage host cells are reported here. The antiparasitic assays have been complemented with enzymatic tests to elucidate the mechanisms of action. New crystal structure description, thermal analysis, magnetic susceptibility and magnetization experiments have also been carried out in order to present a whole characterization of the studied compounds and interesting physical properties besides the biological tests. The results of antiproliferation screening and cytotoxicity show great antiparasitic efficacy in the studied complexes. The superoxide dismutase enzymatic assays exhibit a different behaviour according to the thermochromic triazolopyrimidine form tested. Antiproliferative assays and enzymatic tests corroborate the synergetic leishmanicidal effect present in coordination triazolopyrimidine complexes. The changes in coordination sphere derived from thermochromism affect the physical properties as well as the biological efficacy.

Leishmanicidal and cytotoxic activity from plants used in Tacana traditional medicine (Bolivia)

Etnopharmacological relevanceThirty-eight Tacana medicinal plant species used to treat skin problems, including leishmania ulcers, skin infections, inflammation and wound healing, were collected in the community of Buena Vista, Bolivia, with the Tacana people. Twenty two species are documented for the first time as medicinal plants for this ethnic group living in the northern area of the Department of La Paz. Aim of the studyTo evaluate the leishmanicidal effect (IC50) and cytotoxicity (LD50) of the selected plants. To carry out bioguided studies on the active extracts. To assess the potential of Bolivian plant biodiversity associated with traditional knowledge in the discovery of alternative sources to fight leishmaniasis. Materials and methodsSeventy three ethanol extracts were prepared from 38 species by maceration and were evaluated in vitro against promastigotes of Leishmania amazonensis and L. braziliensis. Active extracts (IC50 ≤ 50 μg/mL) were fractionated by chromatography on Silica gel column and the fractions were assessed against the two Leishmania strains. The most active fractions and the crude extracts were evaluated against reference strains of L. amazonensis, L. braziliensis, L. aethiopica, two native strains (L. Lainsoni and L. braziliensis) and for cytotoxicity against HeLa cells. The chromatographic profile of the active fractions was obtained by reverse phase chromatography using HPLC. ResultsFrom the 73 extracts, 39 extracts (53.4%) were inactive and 34 showed activity. Thirteen species were sselected for bioguided studies. The crude extracts and their 36 fractions were evaluated against two Leishmania strains. The most active fraction were tested in a panel of five leishmania strains and for cytotoxicity. The Selective Index (SI = LD50/IC50) was calculated, and were generally low. Retention time and UV spectra were recorded for the active fractions by HPLC-DAD using a reverse phase column. Profiles were very different from each other, showing the presence of different compounds. ConclusionBolivian traditional knowledge from the Tacanba was useful to identify plants with effect on Leishmania promastigotes. Chromatographic bioguided studies showed stronger leishmanicidal and cytotoxic activity for the medium polar fraction. HPLC analysis showed different chromatographic profiles of the active fractions.

In vitro leishmanicidal and trypanocidal evaluation and magnetic properties of 7-amino-1,2,4-triazolo[1,5-a]pyrimidine Cu(II) complexes

Two triazolopyrimidine complexes have been obtained from reaction between 7-amino-1,2,4-triazolo[1,5-a]pyrimidine (7atp) and Cu (II) salts. Crystal structures of [Cu2(μ-7atp)4Cl2]Cl2·4H2O (1) and [Cu2(μ-7atp)4(H2O)2](NO3)4·H2O (2) have been studied by X-ray diffraction methods and characterized by spectroscopic and thermal analysis. Magnetic studies of these dinuclear complexes have revealed the existence of moderate antiferromagnetic interactions between the copper ions, with J values of −91.2 and −96.1cm−1 respectively. It must be highlighted that the antiparasitic activity of these new complexes has been studied in vitro against three different strains of leishmania spp. and Trypanosoma cruzi, showing a higher efficacy than the 7atp ligand and the reference commercial drugs.

Studies in vitro on infectivity and sensitivity to antileishmanial drugs in New World Leishmania species transfected with the green fluorescent protein [pIR3(-)-eGFP

Current chemotherapeutic agents for leishmaniasis have several disadvantages interfering with the effective treatment and therefore more and better antileishmanial drugs are needed. Discovery of candidates for leishmaniasis treatment requires not only accurate and precise methodologies but also well-known biological system to measure infectivity of parasites and antileishmanial activity of the new compounds. Significant variation in the in vitro and in vivo infectivity and sensitivity to established and experimental drugs in Leishmania strains are reported. This work reports the in vitro biological behavior and antileishmanial drugs sensitivity of different green fluorescent protein transfectant Leishmanias strains. The in vitro growth kinetic and infectivity to U937 cells vary slightly in the Leishmania transfectant strains in comparison with their correspondant wild-type. However, the insertion of the pIR3(-)-eGFP may affect the sensitivity of the parasites to meglumine antimoniate (MA) and miltefosine but not to amphotericin B (AMB) and pentamidine isethionate. In consequence, AMB or pentamidine isethionate but not MA or miltefosine should be used as antileishmanial control drugs during in vitro assays of antileishmanial activity. Furthermore, is recommended to test compounds against more than one Leishmania strain in order to verify that the antileihmanial activity of these compound is similar among species.

Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection by Leishmania (Leishmania) infantum.

A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.

Zoonotic Visceral Leishmania: Modeling and Control

In this work we focus on the transmission dynamics of Visceral strains of leishmania, using mathematical model with two latent compartments in human. From the governing differential equations of the model, we find the reproductive number R0; the number of secondary infection and its biological interpretation. Using Routh- Hurwitz criteria on upper bound matrix, the threshold condition, for stability of the Disease Free State, is calculated. Finally we show that the disease free equilibrium is globally asymptotically stable if R0<<ξ 11.

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

Caseating granulomas in cutaneous leishmaniasis.

BackgroundCaseating granulomas are often associated with a mycobacterial infection (TB) and are thought to be exceedingly rare in cutaneous leishmaniasis (CL). However, no large series has accurately documented the incidence of caseating granulomas in CL.MethodsA multiregional cohort consisting of 317 patients with CL [Syria (157), Pakistan (66), Lebanon (47), Saudi Arabia (43), Ethiopia (2) and Iran (2)] was reviewed. Clinical [age, sex, disease duration, lesion type and geographic and anatomic location] and microscopic data [presence of and type of granuloma, Ridley's parasitic index (PI) and pattern (RP)] were documented. Presence of microorganisms was evaluated using special stains (GMS, PAS, AFB and Gram) and polymerase chain reaction (PCR) for TB and CL. All cases included in this study were confirmed as CL by PCR followed by restriction fragment length polymorphism analysis for molecular speciation and were negative for other organisms by all other studies performed. Categorical and continuous factors were compared for granuloma types using Chi-square, t-test or Mann-Whitney test as appropriate.ResultsGranulomas were identified in 195 (61.5%) cases of CL and these were divided to 49 caseating (25.2%), 9 suppurative (4.6%) and 137 tuberculoid without necrosis (70.2%). Caseating and tuberculoid granuloma groups were significantly different in terms of the geographical source, with more cases harboring caseating granulomas in Saudi Arabia (p<0.0001). Histologically, both groups were also different in the distribution of their RP (p<0.0001) with a doubling RP3 in caseating granulomas (31% vs. 15%) as opposed to doubling of RP5 in tuberculoid granuloma group (38% vs. 19%). Time needed to achieve healing (RP5) was notably shorter in tuberculoid vs. caseating group (4.0 vs. 6.2 months). Parasitic Index, CL species and other considered variables did not differ for the granuloma type groups.ConclusionIn our multiregional large cohort, a notable 18.2% of all CL cases harbored caseating granulomas therefore; CL should be considered part of the differential diagnosis for cases with caseating granulomas in endemic regions, especially considering that the regions included in our cohort are also endemic for TB. Of note, cases of CL with caseating granulomas also showed a slower healing process, with no association with specific species, which may be due to worse host immune response in such cases or to a more aggressive leishmania strains.

Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging

We have set up an assay to study the interactions of live pathogens with their hosts by using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have used this assay to characterize the interactions of Leishmania promastigotes with ~70 mammalian host biomolecules (extracellular proteins, glycosaminoglycans, growth factors, cell surface receptors). We have identified, in total, 27 new partners (23 proteins, 4 glycosaminoglycans) of procyclic promastigotes of six Leishmania species and 18 partners (15 proteins, 3 glycosaminoglycans) of three species of stationary-phase promastigotes for all the strains tested. The diversity of the interaction repertoires of Leishmania parasites reflects their dynamic and complex interplay with their mammalian hosts, which depends mostly on the species and strains of Leishmania. Stationary-phase Leishmania parasites target extracellular matrix proteins and glycosaminoglycans, which are highly connected in the extracellular interaction network. Heparin and heparan sulfate bind to most Leishmania strains tested, and 6-O-sulfate groups play a crucial role in these interactions. Numerous Leishmania strains bind to tropoelastin, and some strains are even able to degrade it. Several strains interact with collagen VI, which is expressed by macrophages. Most Leishmania promastigotes interact with several regulators of angiogenesis, including antiangiogenic factors (endostatin, anastellin) and proangiogenic factors (ECM-1, VEGF, and TEM8 [also known as anthrax toxin receptor 1]), which are regulated by hypoxia. Since hypoxia modulates the infection of macrophages by the parasites, these interactions might influence the infection of host cells by Leishmania.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

A gp63 based vaccine candidate against Visceral Leishmaniasis.

Visceral leishmaniasis is a macrophage associated disorder which leads to a profound decrease in the natural immunotherapeutic potential of the infected subjects to combat the disease. The major surface glycoprotein gp63 has been found to be a significant vaccine candidate against visceral leishmaniasis. The current study addresses the levels of similarity and identity in the gp63 obtained from different species of Leishmania viz donovoni, chagasi and infantum linked to the cause of visceral leishmaniasis. The results from BLAST, Phylogram and Cladogram studies indicate significant identity, similarity and conservation of important residues in the protein which lead us to conclude that a common gp63 based vaccine can be used as a therapeutical tool against visceral leishmaniasis caused by different species strains of leishmania.

Modelling the role of cross-immunity between two different strains of leishmania

Abstract We develop a mathematical model to understand the role of cross-immunity between two different strains of leishmania . Our results show that R 0 , the basic reproduction number of the model decreases as the cross-immunity increases; moreover, if the cross-immunity between the two different strains is perfect, then the disease-free equilibrium is globally asymptotically stable. Results also show that in the case of partial cross-immunity the model undergoes backward bifurcation, where R 0 1 is not enough for eradication of the disease. We find that if the force of infection of both strains in not equal to zero, then both strains will coexist without competition. Numerical results show that at the endemic equilibrium the number of infected humans with one strain is higher than the number of infected humans with the same strain after they recovered from the infection of the other one, which shows that the cross-immunity reduces the number of infections.

Extreme inbreeding in Leishmania braziliensis

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 +/- 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy-Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.

Effect of Leishmania gerbilli Injection on Mice Immunization Against Cutaneous Leishmaniasis (CL) Caused by Leishmania major

In the present study Leishmania gerbilli were used to immunize BALB/c mice against pathogenic strains of leishmania to determine whether injection of L. gerbilli in mice could protect them against later L. major inoculation. Eighty female BALB/c mice were divided by random in eight groups. Promastigotes of L. major and L. gerbilli were used. Mice were inoculated with three different doses of L. gerbilli (3 x 10(6), 2 x 10(7) and 5 x 10(7)) via subcutaneous (SC) in the base of their tails or interpretoen (IP). Forty days after the first injection, all mice received the same doses as a booster. Two control groups received PBS (SC or IP) only. All BALB/c mice were inoculated subcutaneously with 2 x 10(6). Promastigotes of L. major in the base of their tails after 75 days of the first injection of L. gerbilli. When leishmania lesion developed (35 days after challenge), the size was measured and continued once a week for 12 weeks. Meanwhile, the liver and spleen samples of dead mice moved to culture media and examined for the parasite. Delayed Type Hypersensitivity (DTH) and immunoflurecent tests were used to determine results of immunization. Compared with the control group and the other groups that received different doses of L. gerbilli via IP, an evident decrease in lesion size was observed in group that received 2 x 10(7) promastigotes (p < 0.05). By contrast, in those groups received L. gerbilli subcutaneously, no difference was observed through the different doses of inoculated parasite. Comparison of the inoculation styles showed that IP method caused smaller lesions than SC (p < 0.05).

Clonal propagation and the fast generation of karyotype diversity: anin vitro Leishmaniamodel

In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology.

Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis

Three PCR assays for diagnosing leishmaniasis were compared and validated against parasite cultures and microscopic evaluation of stained tissue smears using 92 specimens from suspected cases of cutaneous leishmaniasis (CL) in Israel and the West Bank. Samples from imported and locally acquired disease were examined. The kinetoplast DNA (kDNA) PCR showed the highest sensitivity (98.7%) of any assay, correctly diagnosing 77/78 of the confirmed positive samples, followed by the rRNA gene internal transcribed spacer 1 (ITS1) PCR (71/78 positive, 91.0% sensitivity) and then the spliced leader mini-exon PCR (42/78 positive, 53.8% sensitivity). Either parasite culture or microscopy alone detected 62.8% (49/78) or 74.4% (58/78) of the positive specimens, respectively, while culture and microscopy together improved overall sensitivity to 83.3% (65/78). Except for the kDNA PCR that had six false positives, all other assays were 100% specific. Further, restriction enzyme analysis of the ITS1 PCR product enabled identification of 74.6% of the positive samples, which included strains of Leishmania major (50.9%), Leishmania tropica (47.2%), and the Leishmania braziliensis complex (1.9%). This suggests that a PCR using kDNA should be used for the diagnosis of CL and that an ITS1 PCR can be reliably used for the diagnosis of CL when rapid species identification is needed.

Multilocus Microsatellite Typing as a New Tool for Discrimination of Leishmania infantum MON-1 Strains

The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.

A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene.

BackgroundLeishmaniases are among the most proteiform parasitic infections in humans ranging from unapparent to cutaneous, mucocutaneous or visceral diseases. The various clinical issues depend on complex and still poorly understood mechanisms where both host and parasite factors are interacting. Among the candidate factors of parasite virulence are the A2 genes, a family of multiple genes that are developmentally expressed in species of the Leishmania donovani group responsible for visceral diseases (VL). By contrast, in L. major determining cutaneous infections (CL) we showed that A2 genes are present in a truncated form only. Furthermore, the A2 genomic sequences of L. major were considered subsequently to represent non-expressed pseudogenes [1]. Consequently, it was suggested that the structural and functional properties of A2 genes could play a role in the differential tropism of CL and VL leishmanias. On this basis, it was of importance to determine whether the observed structural/functional particularities of the L. major A2 genes were shared by other CL Leishmania, therefore representing a proper characteristic of CL A2 genes as opposed to those of VL isolates.MethodsIn the present study we amplified by PCR and sequenced the A2 genes from genomic DNA and from clonal libraries of the four Old World CL species comparatively to a clonal population of L. infantum VL parasites. Using RT-PCR we also amplified and sequenced A2 mRNA transcripts from L. major.ResultsA unique A2 sequence was identified in Old World cutaneous Leishmania by sequencing. The shared sequence was highly conserved among the various CL strains and species analysed, showing a single polymorphism C/G at position 58. The CL A2 gene was found to be functionally transcribed at both parasite stages.ConclusionThe present study shows that cutaneous strains of leishmania share a conserved functional A2 gene. As opposed to the multiple A2 genes described in VL isolates, the CL A2 gene is unique, lacking most of the nucleotide repeats that constitute the variable region at the 5'end of the VL A2 sequences. As the variable region of the VL A2 gene has been shown to correspond to a portion of the protein which is highly immunogenic, the present results support the hypothesis of a possible role of the A2 gene in the differential tropism of CL and VL leishmania parasites.