Low Plasma Lipids Are Associated with Relapsing and Lethal Visceral Leishmaniasis in HIV-Infected Patients.

Visceral leishmaniasis (VL) results from protozoa Leishmania infantum and L. donovani infection. This study investigated whether host factors would explain the relapses. First, susceptibility to amphotericin B of L. infantum isolates was evaluated in vitro. Then, clinical data and the lipid profile of patients with relapsing and non-relapsing VL were assessed. Susceptibility to amphotericin B was similar between the isolates. CD4+ lymphocytes were reduced in both groups of patients in the first episode and with relapsing VL. Still, the strongest blood cell indicator associated with relapses was low total lymphocyte counts. Total plasma cholesterol, high-density lipoprotein, low-density lipoprotein, and, uniquely, triglycerides of the six individuals in the first episode and twenty-three with relapsing VL were lower in relapsing patients than those in the first episode. Deceased patients had extremely low low-density lipoprotein. After CD4+ decreases, lymphocyte CD8+ reduction is the final stage of immunological failure. The lower lipid concentrations appear to be secondary to the depletion of fat stores by inflammation-induced cachexia and fat exhaustion provoked by the co-occurrence of both diseases, which can finally lead to death.

Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein­protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.

The Extraordinary Case of a Woman with a 30-Year-Long Diffuse Leishmaniasis Cured with One Single Ampoule of Intranasal Pentavalent Antimoniate.

Infection with Leishmania amazonensis and L. mexicana may lead to diffuse cutaneous leishmaniasis. The cure is exceptional, especially for the strange case of this lady. Case report: The patient acquired the disease in childhood and remained with lesions for over 30 years, albeit several treatments. She worsened after a pregnancy, developing disseminated lesions. Miltefosine with amphotericin B and pentamidine resulted in remission. Lesions reappeared after one year, accompanied by intra-nasal infiltration of the disease. The nasal spraying of a single ampoule of pentavalent antimoniate resulted in the sustained disappearance of the nasal symptoms and all the cutaneous lesions. After over eight years, she remains disease-free, albeit under renal replacement therapy. The high nasal mucosal antimonial concentration may explain the long-lasting cure via new MHC class I epitope-specific CD8+ cell clones against L. amazonensis present in the nasal mucosa.

In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses.

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Repurposing topical triclosan for cutaneous leishmaniasis: Preclinical efficacy in a murine Leishmania (L.) amazonensis model.

Leishmaniasis remains an important neglected tropical infection caused by the protozoan Leishmania and affects 12 million people in 98 countries. The treatment is limited with severe adverse effects. In the search for new therapies, the drug repositioning and combination therapy have been successfully applied to neglected diseases. The aim of the present study was to evaluate the in vitro and in vivo anti-Leishmania (Leishmania) amazonensis potential of triclosan, an approved topical antimicrobial agent used for surgical procedures. in vitro phenotypic studies of drug-treated parasites were performed to evaluate the lethal action of triclosan, accompanied by an isobolographic ex-vivo analysis with the association of triclosan and miltefosine. The results showed that triclosan has activity against L. (L.) amazonensis intracellular amastigotes, with a 50% inhibitory concentration of 16 µM. By using fluorescent probes and transmission electron microscopy, a pore-forming activity of triclosan toward the parasite plasma membrane was demonstrated, leading to depolarization of the mitochondrial membrane potential and reduction of the reactive oxygen species levels in the extracellular promastigotes. The in vitro interaction between triclosan and miltefosine in the combination therapy assay was classified as additive against intracellular amastigotes. Leishmania-infected mice were treated with topical triclosan (1% base cream for 14 consecutive days), and showed 89% reduction in the parasite burden. The obtained results contribute to the investigation of new alternatives for the treatment of cutaneous leishmaniasis and suggest that the coadministration of triclosan and miltefosine should be investigated in animal models.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the ß-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis ß-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major ß-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) ß-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

Cellular infiltrate in cutaneous leishmaniasis lesions and therapeutic outcome

Abstract Background: The treatment of cutaneous leishmaniasis is a challenge. A better understanding of the in situ mechanisms involved in the evolution and cure of the disease is essential for the development of new therapies. Objective: Correlate histopathological and immunological characteristics of cutaneous leishmaniasis lesions with clinical outcome after different treatment regimens. Methods: The authors analyzed cellular infiltration and immunohistochemistry staining for CD4, CD8 and IL-17 in biopsy samples from 33 patients with cutaneous leishmaniasis before treatment. All patients were recruited in a randomized clinical trial at Corte de Pedra (Bahia-Brazil) and assigned to receive Glucantime®, Glucantime® + Oral Tamoxifen or Glucantime® + Topical Tamoxifen. Patients were followed for 2 to 6 months to define disease outcome. Results: A similar expression of CD4, CD8 and IL-17 was observed in lesion samples regardless of clinical outcome. In general, a higher amount of CD8 cells were observed compared with CD4 cells. An important observation was that all patients whose cellular infiltrate did not contain plasma cells were cured after treatment. Study limitations: Isolated quantification of TCD8 and IL-17 using immunohistochemistry is insufficient to analyze the role of these molecules in the immunopathogenesis of cutaneous leishmaniasis. In addition, the expansion of the immunohistochemistry panel would allow a more complete analysis of the immune response in situ. Conclusions: The absence of plasma cells in cutaneous leishmaniasis lesions was related to a favorable therapeutic outcome.

Full nucleotide sequencing of ribosomal DNA internal transcribed spacer of Leishmania species causing cutaneous leishmaniasis in Brazil and its potential for species typing.

Species-specific diagnosis still represents a challenge in leishmaniasis management, particularly in regions with multiple endemic species. In Brazil, seven species have been recognized as etiological agents of cutaneous leishmaniasis. The disease comprises complex clinical presentation patterns, classified as localized, diffuse, disseminated and mucocutaneous leishmaniasis. In this study, we characterized the full nucleotide sequence of a region comprising the ribosomal DNA internal transcribed spacers 1 and 2 and 5.8 S gene of reference strains of Leishmania (Viannia) species reported as causative agents of human leishmaniasis in Brazil. The analysis of the nucleotide sequence of this region was able to discriminate species in the Leishmania (Viannia) subgenus and to determine intra- and interspecies phylogenetic relationships.

In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil.

Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 µM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 µM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

Cellular infiltrate in cutaneous leishmaniasis lesions and therapeutic outcome.

BACKGROUND: The treatment of cutaneous leishmaniasis is a challenge. A better understanding of the in situ mechanisms involved in the evolution and cure of the disease is essential for the development of new therapies. OBJECTIVE: Correlate histopathological and immunological characteristics of cutaneous leishmaniasis lesions with clinical outcome after different treatment regimens. METHODS: The authors analyzed cellular infiltration and immunohistochemistry staining for CD4, CD8 and IL-17 in biopsy samples from 33 patients with cutaneous leishmaniasis before treatment. All patients were recruited in a randomized clinical trial at Corte de Pedra (Bahia-Brazil) and assigned to receive Glucantime®, Glucantime® + Oral Tamoxifen or Glucantime® + Topical Tamoxifen. Patients were followed for 2 to 6 months to define disease outcome. RESULTS: A similar expression of CD4, CD8 and IL-17 was observed in lesion samples regardless of clinical outcome. In general, a higher amount of CD8 cells were observed compared with CD4 cells. An important observation was that all patients whose cellular infiltrate did not contain plasma cells were cured after treatment. STUDY LIMITATIONS: Isolated quantification of TCD8 and IL-17 using immunohistochemistry is insufficient to analyze the role of these molecules in the immunopathogenesis of cutaneous leishmaniasis. In addition, the expansion of the immunohistochemistry panel would allow a more complete analysis of the immune response in situ. CONCLUSIONS: The absence of plasma cells in cutaneous leishmaniasis lesions was related to a favorable therapeutic outcome.

Ros3 (Lem3p/CDC50) Gene Dosage Is Implicated in Miltefosine Susceptibility in Leishmania (Viannia) braziliensis Clinical Isolates and in Leishmania (Leishmania) major.

The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine.

2-aminobenzimidazoles for leishmaniasis: From initial hit discovery to in vivo profiling.

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 µM) and 39 (L. infantum IC50: 0.5 µM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency.

Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model.

New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis.

Route map for the discovery and pre-clinical development of new drugs and treatments for cutaneous leishmaniasis.

Although there have been significant advances in the treatment of visceral leishmaniasis (VL) and several novel compounds are currently in pre-clinical and clinical development for this manifestation of leishmaniasis, there have been limited advances in drug research and development (R & D) for cutaneous leishmaniasis (CL). Here we review the need for new treatments for CL, describe in vitro and in vivo assays, models and approaches taken over the past decade to establish a pathway for the discovery, and pre-clinical development of new drugs for CL. These recent advances include novel mouse models of infection using bioluminescent Leishmania, the introduction of PK/PD approaches to skin infection, and defined pre-clinical candidate profiles.

Investigation of the pathways related to intrinsic miltefosine tolerance in Leishmania (Viannia) braziliensis clinical isolates reveals differences in drug uptake.

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania.

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48 µM (95% CI 7.68-9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion.

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.

Case Report: Atypical Cutaneous Leishmaniasis in a Patient with Mixed Leishmania guyanensis and Leishmania amazonensis Infection.

The disseminated form of leishmaniasis is a serious and rare disease, being diagnosed in 2% of the cutaneous cases registered per year in Brazil. The main characteristic is the appearance of multiple pleomorphic lesions on the cutaneous surface. A 68-year-old male from the rural area of Tocantins, Brazil, presented atypical disseminated cutaneous leishmaniasis (ACL). The clinical course and histopathological and immunological findings presented a mixed pattern that hindered diagnosis and therapeutic management. Molecular typing revealed a mixed infection with Leishmania (V.) guyanensis and Leishmania (L.) amazonensis. Molecular identification of the agents responsible for ACL is important for adequate therapeutic planning, minimizing the possibility of sequellae that impact the quality of life of the patient.

Tamoxifen and meglumine antimoniate combined therapy in cutaneous leishmaniasis patients: a randomised trial.

OBJECTIVES: There is a clear need for new strategies of leishmaniasis treatment. This work was conducted to evaluate the efficacy of the co-administration of tamoxifen and meglumine antimoniate (SbV ) in a phase II pilot clinical trial in localised cutaneous leishmaniasis patients. METHODS: A randomised controlled pilot clinical trial was conducted to evaluate the efficacy and safety of oral (40 mg/day for 20 days) or topical tamoxifen (0.1% tamoxifen citrate for 20 days) combined with meglumine antimoniate (20 mg SbV /kg/day for 20 days) vs. a standard SbV protocol (20 mg/kg/day for 20 days) for the treatment of cutaneous leishmaniasis. Primary outcome was complete epithelisation of the lesion 6 months after the end of treatment. Secondary outcomes were lesion healing 2 months after the end of treatment and frequency and severity of adverse events. RESULTS: A total of 38 subjects were included in the trial, 15 were treated with standard SbV and 23 with the combination of tamoxifen and SbV . Of the patients treated with the co-administration scheme, 12 received tamoxifen orally and 11 were treated with topical tamoxifen. Tamoxifen administered by the oral or topical routes was well tolerated. Cure rates 6 months after the end of treatment per intention to treat were 40% in the group treated with the standard SbV scheme, and 36.4% and 58%, respectively, for groups treated with SbV plus topical or oral tamoxifen. CONCLUSIONS: In the doses and schemes used in this study, co-administration of oral tamoxifen and SbV resulted in higher cure rates in comparison with the standard scheme of treatment, although not to statistically significant levels.

Identification of Leishmania (Viannia) species and clinical isolates of Leishmania (Leishmania) amazonensis from Brazil using PCR-RFLP of the heat-shock protein 70 gene reveals some unexpected observations.

Hsp70 is a cytoplasmic heat-shock protein, encoded by a multicopy tandemly repeated gene that has recently been gaining popularity as a valuable marker for typing Leishmania species. In this study, we used a previously described hsp70 PCR-RFLP method for identifying Brazilian Leishmania isolates. We identified two distinct L. (L.) amazonensis hsp70 alleles that resulted in two different RFLP patterns. Also, we found RFLP polymorphisms amongst L. (Viannia) naiffi strains. The profiles of both L. (V.) shawi and L. (V.) lindenbergi were very similar to those of other L. (Viannia) species. The observations described herein reflect the polymorphism found within species of Leishmania and indicate that results from this hsp70 PCR-RFLP method should be used with caution when typing isolates from clinical cases of leishmaniasis and Leishmania species from Brazil.