Search

We showed previously that antioxidant enzyme heme oxygenase 1 (HO-1) is critical for Leishmania survival in visceral leishmaniasis. HO-1 inhibits host oxidative burst and inflammatory cytokine production, leading to parasite persistence. In the present study, screening of reported HO-1 transcription factors revealed that infection upregulated (4.1-fold compared to control [ P  < 0.001]) nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2). Silencing of NRF2 reduced both HO-1 expression and parasite survival. Investigation revealed that infection-induced transient reactive oxygen species (ROS) production dissociated NRF2 from its inhibitor KEAP1 and enabled phosphorylation-dependent nuclear translocation. Both NRF2 and HO-1 silencing in infection increased production of proinflammatory cytokines. But the level was greater in NRF2-silenced cells than in HO-1-silenced ones, suggesting the presence of other targets of NRF2. Another stress responsive transcription factor ATF3 is also induced (4.6-fold compared to control [ P  < 0.001]) by NRF2 during infection. Silencing of ATF3 reduced parasite survival (59.3% decrease compared to control [ P  < 0.001]) and increased proinflammatory cytokines. Infection-induced ATF3 recruited HDAC1 into the promoter sites of tumor necrosis factor alpha (TNF-α) and interleukin 12b (IL-12b) genes. Resulting deacetylated histones prevented NF-κB promoter binding, thereby reducing transcription of inflammatory cytokines. Administering the NRF2 inhibitor trigonelline hydrochloride to infected BALB/c mice resulted in reduced HO-1 and ATF3 expression, decreased spleen and liver parasite burdens, and increased proinflammatory cytokine levels. These results suggest that Leishmania upregulates NRF2 to activate both HO-1 and ATF3 for disease progression.

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon's, Faith's and Pielou's indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales&#95;uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally., Competing Interests: The authors have declared that no competing interests exist.

In our interest in oxabicyclic compounds as potent antileishmanial agents, the present work deals with the chemical synthesis of a new oxabicyclic derivative, methyl 4-(7-hydroxy-4,4,8-trimethyl-3-oxabicyclo[3.3.1]nonan-2-yl)benzoate (PS-207). This oxabicyclic derivative showed a good antileishmanial effect on the parasite, on both the promastigote and the amastigote. The mode of parasitic death from PS-207 seemed to be apoptosis-like. Interestingly, the combination of PS-207 with a low dose of miltefosine showed a synergistic effect against the parasite., (Copyright © 2018 American Society for Microbiology.)

In Morocco, Leishmania infantum is the main etiologic agent of human and canine visceral leishmaniasis (VL). This species has been proven to be an opportunistic agent in HIV+ patients and is also responsible of sporadic cutaneous leishmaniasis (CL).This work aims to evaluate the genetic variability of Moroccan L. infantum strains based on PCR-RFLP analysis of the kinetoplastid DNA (kDNA) minicircles. A total of 75 DNA samples extracted from positive Giemsa-stained smears (n=32) and from L. infantum cultures (n=43) was studied. The samples have been taken from VL patients infected (n=7) or not (n=56) by HIV, patients with CL (n=2) and finally from infected dogs (n=10). An hypervariable region of kDNA was amplified using the primers MC1 and MC2; the PCR products were digested separately by a panel of nine restriction enzymes. The presence or absence of restriction fragments was scored in a binary matrix and the SplitsTree4 software was used for the construction of a Neighbor-Net network. Moroccan L. infantum population showed an important level of variability with the identification of 6 genotypes. For each genotype a PCR product was sequenced, confirming the presence of all the expected restriction sites. The predominant profile was the genotype B. A new genotype, named Q was detected for the first time, whereas the four other genotypes (G, K, N and O) were reported sporadically in the Mediterranean basin. The Neighbor-Net network segregates our L. infantum population into 3 clusters: Cluster I includes genotype B, cluster II grouping the genotypes O, Q and G and finally the cluster III contains the genotype N. The kDNA-PCR-RFLP assay is suitable for use directly on biological samples; it reveals an important degree of genetic variability among L. infantum strains even those belonging to the same zymodeme what is of great epidemiological interest., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A recent report on the taxonomic profile of the human gut microbiome in pre-Columbian mummies (Santiago-Rodriguez et al. 2016) gives for the first time evidence of the presence of Leishmania DNA (sequences similar to Leishmania donovani according to the authors) that can be reminiscent of visceral leishmaniasis during the pre-Columbian era. It is commonly assumed that Leishmania infantum, the etiological agent of American visceral leishmaniasis (AVL) was introduced into the New World by the Iberian conquest. This finding is really surprising and must be put into perspective with what is known from an AVL epidemiological and historical point of view. Beside L. infantum, there are other species that are occasionally reported to cause AVL in the New World. Among these, L. colombiensis is present in the region of pre-Columbian mummies studied. Other explanations for these findings include a more ancient introduction of a visceral species of Leishmania from the Old World or the existence of a yet unidentified endemic species causing visceral leishmaniasis in South America. Unfortunately, very few molecular data are known about this very long pre-Columbian period concerning the circulating species of Leishmania and their diversity in America., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The incidence of visceral leishmaniasis (VL) after solid organ transplantation (SOT) is increasing. The optimal therapy for post-transplant VL remains unclear, as relapses after liposomal amphotericin B (L-AmB) are common. Miltefosine has been shown to be effective for treating VL in immunocompetent patients, although data in the specific population of SOT recipients are lacking. In the setting of an outbreak of leishmaniasis occurring in Southwest Madrid, we reviewed our experience in 6 SOT recipients with persistent or relapsing VL who received a 28-day course of miltefosine (2.5 mg/kg/day) as salvage therapy. All patients had been treated previously with L-AmB as first-line therapy. The incident episode of VL occurred at a median of 14 months after transplantation. Two patients experienced persistent infection and the remaining 4 had a relapse after a median interval of 168 days since the completion of the course of L-AmB. All the patients had an apparent initial clinical improvement with miltefosine. However, VL relapsed in 3 of them (after a median interval of 46 days), which required retreatment with L-AmB-based regimens. Miltefosine therapy was followed by a prolonged secondary prophylaxis with L-AmB in the only 2 cases with sustained clinical response and ongoing immunosuppression. No adverse effects associated with miltefosine were observed. Albeit limited, our experience suggests that miltefosine monotherapy likely has a limited utility to obtain a long-lasting clinical response in complicated (persistent or relapsing) forms of post-transplant VL, although its role in association with L-AmB-based secondary prophylaxis may merit further investigation., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), a heterogeneous population of precursor cells, modulate protective immunity against visceral leishmaniasis by suppressing T cell functions. We observed that CD11b(+) Gr1(+) MDSCs, which initially expanded in soluble leishmanial antigen (SLA)-immunized mice and later diminished, suppressed proliferation of T cells isolated from SLA-immunized mice, but to a lesser extent than the case in naive mice. This lesser suppression of MDSCs accompanied the expression of F4/80 and the production of Cox-2, arginase I, nitric oxide, and PGE2. However, with SLA immunization, there was no difference in the expression of interleukin-2 (IL-2) or gamma interferon (IFN-γ) by T cells, in contrast to the case in nonimmunized mice, in which there is an influence. Glycyrrhizic acid (a triterpenoid compound)-mediated inhibition of Cox-2 in myeloid-derived suppressor cells influenced the capacity of T cells to proliferate and the expression of IL-2 and IFN-γ in Leishmania donovani-infected BALB/c mice. Further characterization confirmed that administration of glycyrrhizic acid to L. donovani-infected BALB/c mice results in an impairment of the generation of MDSCs and a reciprocal organ-specific proliferation of IFN-γ- and IL-10-expressing CD4(+) and CD8(+) T cells. Comprehensive knowledge on the Cox-2-mediated regulation of myeloid-derived suppressor cells might be involved in unlocking a new avenue for therapeutic interventions during visceral leishmaniasis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Using flow cytometry, we evaluated the frequencies of CD4(+) and CD8(+) T cells and Foxp3(+) regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4(+), total Foxp3, and CD4(+) Foxp3(+) cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-β, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-β, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.

Novel antileishmanials are urgently required to overcome emergence of drug resistance, cytotoxic effects, and difficulties in oral delivery. Toward this, we investigated a series of novel 4-aminoquinaldine derivatives, a new class of molecules, as potential antileishmanials. 4-Aminoquinaldine derivatives presented inhibitory effects on L. donovani promastigotes and amastigotes (50% inhibitory concentration range, 0.94 to 127 μM). Of these, PP-9 and PP-10 were the most effective in vitro and demonstrated strong efficacies in vivo through the intraperitoneal route. They were also found to be effective against both sodium antimony gluconate-sensitive and -resistant Leishmania donovani strains in BALB/c mice when treated orally, resulting in more than 95% protection. Investigation of their mode of action revealed that killing by PP-10 involved moderate inhibition of dihydrofolate reductase and elicitation of the apoptotic cascade. Our studies implicate that PP-10 augments reactive oxygen species generation, evidenced from decreased glutathione levels and increased lipid peroxidation. Subsequent disruption of Leishmania promastigote mitochondrial membrane potential and activation of cytosolic proteases initiated the apoptotic pathway, resulting in DNA fragmentation and parasite death. Our results demonstrate that PP-9 and PP-10 are promising lead compounds with the potential for treating visceral leishmaniasis (VL) through the oral route.

Background: Leishmaniasis includes a spectrum of diseases caused by protozoan parasites belonging to the genus Leishmania. The disease is traditionally classified into visceral, cutaneous, or mucocutaneous leishmaniasis, depending on clinical characteristics as well as the species involved. Leishmania tropica is one of the causative agents of cutaneous leishmaniasis, with a typical incubation period of weeks to months. Observation We describe a 17-year-old Afghani girl who had lived in the United States for 4 years and who presented with a 6-month history of pretibial ulcerations, 9.1-kg weight loss, abdominal pain, splenomegaly, and extreme fatigue. Histopathologic examination and culture with isoenzyme electrophoresis speciation of her skin lesions confirmed the presence of L tropica. In addition, results of serum laboratory and serological studies were highly suggestive of concomitant visceral involvement. The patient was treated with a 28-day course of intravenous pentavalent antimonial compound sodium stibogluconate with complete resolution of her systemic signs and symptoms and improvement of her pretibial ulcerations., Conclusions: This is an exceptional case in that our patient presented with disease after an incubation period of years rather than the more typical weeks to months. In addition, this patient had confirmed cutaneous involvement, as well as strong evidence of viscerotropic disease caused by L tropica, a species that characteristically displays dermotropism, not viscerotropism.

Background: Heat shock protein 70 (HSP70) is present in all organisms studied so far, and is a major immunogen in infections caused by pathogens including Leishmania spp., Objective: The aim of this study was to clone and express HSP70 from L. infantum strain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceral leishmaniasis (VL)., Methods: The L. infantum HSP70 gene segment was amplified by specific primers. It was cloned into pTZ57R vector and subcloned into pET32a (+) expression vector. The new construct was transformed in the E.coli Rosetta strain, and HSP70 protein was expressed in the presence of 1 mM IPTG and purified using a HiTrap chelating column. Antibody responses against HSP70 were determined by ELISA in 37 patients with visceral leishmaniasis and 63 healthy controls., Results: Expression of HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with an anti-His tag antibody. There was no difference between the sequence of nucleotides of the HSP70 gene in the present study and other reported sequences. The ELISA results indicated that the sera of 81.1% (30/37) of the patients and 6.3% (5/63) of controls reacted to L. infantum HSP70., Conclusion: The conservative nature of the HSP70 molecule is an advantage in vaccine studies, because of minor differences (6%) between the nucleotide sequences and consequently the similarity in amino acid sequences in various strains of L. infantum. It could therefore be used in vaccine research against leishmaniasis and also as a tool for serodiagnosis.

Three novel diagnostic tests for visceral leishmaniasis (VL), namely FD-DAT, rK39 dipstick and KATEX, were evaluated under field conditions using 101 clinical cases suspected of having VL enrolled in a trial either by active (63 patients) or passive (38 patients) surveillance. VL was confirmed in 49 patients: 35 by both aspirate smear microscopy and NNN culture, 10 by NNN culture alone and 4 by aspirate smear microscopy alone. Based on tests performed in the field, sensitivity for FD-DAT, rK39 dipstick and KATEX was determined to be 95.3% (95% CI 82.9-99.2%), 71.7% (95% CI 56.3-83.5%) and 57.4% (95% CI 42.3-71.4%), respectively. Similarly, the specificity was determined to be 62.7% (95% CI 48.1-75.5%), 82.4% (95% CI 68.6-91.1%) and 84.3% (95% CI 70.9-92.5%), respectively. A higher sensitivity of KATEX (73.9% vs. 41.7%) and higher specificity of FD-DAT (100.0% vs. 48.6%) were demonstrated under passive case detection compared with active case detection. FD-DAT is recommended for confirmation of VL diagnosis in hospital settings, whereas its use in the field will be limited to exclude VL in clinical suspects. The sensitivity of KATEX and rK39 dipstick tests needs to be improved to promote their use as first-line diagnostic tests in the field setting of northwestern Ethiopia.

Solute carrier family 11a member 1 (Slc11a1; formerly natural resistance-associated macrophage protein 1) encodes a late endosomal/lysosomal protein/divalent cation transporter, which regulates iron homeostasis in macrophages. During macrophage activation, Slc11a1 exerts pleiotropic effects on gene regulation and function, including generation of nitric oxide (NO) via inducible NO synthase (iNOS; encoded by Nos2A) and of reactive oxygen intermediates (ROI) via the phagocyte oxidase complex. As NO and ROI have potent antimicrobial activity in macrophages, it was assumed that their activities would contribute to Slc11a1-regulated innate resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani. By intercrossing mice with gene disruptions at Nos2A and Cybb (encoding gp91phox, the heavy chain subunit of cytochrome b-245 and an essential component of phagocyte NADPH oxidase) onto equivalent Slc11a1 wild-type and mutant genetic backgrounds, we demonstrate that neither iNOS nor gp91phox activity is required for Slc11a1-mediated innate resistance to either infection. Functional gp91phox and iNOS are required to control S. enterica serovar Typhimurium in non-Slc11a1-regulated phases of infection. For L. donovani, an organ-specific requirement for iNOS to clear parasites from the spleen was observed at 50 days post-infection, but neither iNOS nor gp91phox influenced late-phase infection in the liver. This contrasted with Leishmania major infection, which caused rapid lesion growth and death in iNOS knockout mice and some exacerbation of disease with gp91phox deficiency. This highlights the adaptive differences in tissue and cellular tropisms between L. donovani and L. major and the different genes and mechanisms that regulate visceral versus cutaneous forms of the disease.

Hepatic tissue samples were obtained from 26 dogs humanely destroyed because of naturally occurring leishmaniosis (Leishmania infantum). None of the animals had palpable hepatomegaly or any other physical finding or historical evidence indicative of liver failure. However, serum biochemistry revealed hypoalbuminaemia (6/26), increased alkaline phosphatase (ALP) activity (15/26), and increased concentrations of total bilirubin (2/26) and post-prandial bile acids (4/26). Three main histological patterns were identified. In pattern 1 (3/26), the liver microarchitecture remained unchanged apart from the presence of individual or clustered macrophages in the sinusoids. In pattern 2 (20/26), there was multifocal, mild to moderate, granulomatous to pyogranulomatous infiltration of the hepatic parenchyma, particularly in the portal areas. Pattern 3 (3/26), which was the most severe form, was characterized by marked portal lymphoplasmacytic infiltration with occasional broaching of the limiting plate and extension into the adjacent parenchyma. In this pattern there was also mild portal fibrosis, together with lymphoplasmacytic aggregates within the parenchyma and small clusters of lymphocytes and plasma cells within the sinusoids. All three patterns were associated with hepatocyte vacuolation (15/26 dogs), and haemosiderin accumulation within the hepatocyte cytoplasm. Congestion was present in the liver of five dogs. No correlation was found between histopathological pattern and breed, sex, age, clinical manifestations, serum biochemical profile or parasite load in the hepatic tissue; patterns 1-3 may, however, represent sequential stages of hepatic leishmania infection during the chronic course of the disease.

Visceral leishmaniasis (VL) or kala-azar (KA) affects the rural poor, causing significant morbidity and mortality. We examined the epidemiological and social impact of KA in an affected village in Bangladesh. A population-based survey of the village residents showed a case fatality rate of 14.7% among females and 5.3% among males. Before initiation of the study, female patients were ill longer than males before they received treatment. Future work needs to focus on understanding the implications of KA on women and to develop sustainable strategies for appropriate and timely access to treatment.

Two forms of cutaneous leishmaniasis are endemic in Algeria. Zoonotic cutaneous leishmaniasis caused by Leishmania major is widespread in the steppe regions of the northern Sahara. Sporadic cutaneous leishmaniasis caused by Leishmania infantum occurs in the north along the coastline. The main causative agent for the sporadic form is Leishmania infantum zymodeme MON-24. It has been isolated from the sandfly vector Phlebotomus perfiliewi. However all attempts to isolate this variant from dogs, i.e., the main reservoir of Leishmania infantum, have failed. The authors report the first successful detection of Leishmania infantum MON-24 in the dog.

A total of 51 inpatients having prolonged low grade irregular fever with anaemia, hepatosplenomegaly, emaciation and other allied features were evaluated by findings of haematologic, seroimmunologic, microscopic examination for LD body and culture for L. donovani. The study was done during the period from September' 1999 to January' 2000. Bone marrow or splenic aspirates were examined for LD bodies and those samples were cultured in modified NNN media following a standard method. Out of 51 samples, 36 (70.5%) were positive for LD bodies in stained smears and 38 (74.5%) were positive in culture. The mean time of culture positivity was 5 +/- 2 days. Specific antileishmanial antibody were detected by Immunochromatography Test (ICT) in all 38 confirmed cases, whereas Aldehyde test (AT) were negative in 3 such cases. ICT was positive in 1 and AT in 7 cases where no parasite could be detected. So, sensitivity of ICT was 100% with a specificity of 92.3% and sensitivity of AT was 92.1% with a specificity of 46.1%.

The visceral form of leishmaniasis caused by Leishmania infantum is frequently observed in Mediterranean countries, however otorhinolaryngeal involvement has only rarely been reported in the literature. We report here the case of a 23-year-old Cameroon man, resident in France since 1991, and presenting with recurrent epistaxis and an epiglottis tumoral lesion. The visceral form of leishmaniasis with hepatosplenomegaly and pancytopenia was diagnosed. Culture of the epiglottal lesion revealed L. infantum. Serological test for human immunodeficiency virus was negative and the patient was not on immunosuppressive drugs. Dramatic improvement was observed with antimony.

The variable and conserved sequence boxes of kinetoplast DNA (kDNA) of 11 standard strains of 6 complexes of New and Old World Leishmania were amplified using PCR. Four strains from 2 complexes of Old World Leishmania - L. major (MRHO/IR/64/Nadim-1), with 2 bands at 850 and 620 bp, L. major (MHOM/SU/73/5-ASKH), with a band at 620 bp, L. donovani, with a band at 800 bp and L. infantum, with a band at 650 bp - could be differentiated from each other and from the New World strains, with the exception of L. infantum. Seven Leishmania strains from 4 complexes of New World Leishmania - L. mexicana and L. pifanoi, with a band at 730 bp, L. guyanensis, with 2 bands at 730 and 650 bp, L. peruviana, with a band at 710 bp and L. amazonensis, L. garnhami and L. braziliensis, each with a band at 650 bp - were identified. Of these strains, L. guyanensis and L. peruviana could be differentiated from each other and from the Old World strains. These results show that using PCR amplification of kDNA we could differentiate between New and Old World Leishmania at both complex and strain levels. The amplified kDNA PCR products, together with other techniques, could be useful as a diagnostic tool for the identification of Leishmania species.