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In 2022, the Pan American Health Organization recommended the intralesional application (IL) of pentavalent antimonials in adult patients with localized cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis. Other guidelines differ from that recommendation, considering that infections caused by a Leishmania species that can be associated with increased risk for mucosal leishmaniasis, in particular L. (V.) braziliensis, should not be eligible for intralesional treatment. This was a prospective interventional study carried out with eight patients diagnosed with CL residing in northeast Brazil during the period from 2019 to 2022. To our knowledge, this is the first prospective study on the subject conducted in the northeast region, which has the second-highest number of cases in the country. In our sample, clinical cure was achieved with the use of intralesional treatment in all cases, and there were no serious adverse events or mucosal involvement during the 1-year follow-up period. We emphasize the importance of using the right criteria for choosing this therapeutic modality and highlight the advantages of intralesional treatment due to the lower risk of adverse events and cost reduction to health services.

Outbreaks of cutaneous leishmaniasis are relatively common among soldiers involved in nocturnal activities in tropical forests. We investigated the population dynamics of sand flies in a military training camp located in a remnant of Atlantic rainforest in northeastern Brazil, where outbreaks of cutaneous leishmaniasis have sporadically been described. From July 2012 to July 2014, light traps were monthly placed in 10 collection sites, being nine sites located near the forest edge and one near a sheep and goat stable. Light traps operated from 5:00 pm to 6:00 am, during four consecutive nights. Leishmania infection in sand flies was assessed using a fast real-time PCR assay. Cases of cutaneous leishmaniasis among soldiers were also investigated. In total, 24,606 sand flies belonging to 25 species were identified. Males (n = 12,683) predominated over females (n = 11,923). Sand flies were present during all months, being more numerous in March (n = 1,691) and April 2013 (n = 3,324). Lutzomyia choti (72.9%) was the most abundant species, followed by Lutzomyia longispina (13.8%), Lutzomyia complexa (5.3%), representing together >90% of the sand flies collected. Forty cases of cutaneous leishmaniasis were recorded among soldiers from January 2012 to December 2014. Leishmania isolates were obtained from eight patients and were all characterized as Leishmania braziliensis. Soldiers and anyone overnighting in Atlantic rainforest remnants should adopt preventative measures such as the use of repellents on bare skin or clothes and insecticide-treated tents.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL., (Copyright © 2017 Sato et al.)

The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals.

Background: Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques., Methods: qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains., Results: Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing and qPCR (p = 0.2566). In contrast, a highly significant difference was observed for qPCR and RFLP (p < 0.001)., Conclusions: In this study, we demonstrated the potential use of qPCR as a tool for Leishmania species identification using two Tm ranges., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The outcome of Leishmania infections varies substantially, depending on the host and the parasite strain; infection may be asymptomatic or cause mild or severe skin ulcers (cutaneous leishmaniasis [CL]), limited or disseminated lesions, or lethal visceral disease. We previously reported an association between IL-2R mutations and susceptibility to visceral leishmaniasis in children infected with Leishmania donovani. In the present study, we evaluated the possible role of IL-2 signaling in human CL. We first showed that the transcripts of several genes of the IL-2 pathway were abundant in skin lesions caused by Leishmania braziliensis. We then carried out a genetic analysis, focusing on major genes of the IL-2 pathway. We used a family-based approach and found that polymorphisms of several genes appeared to be associated with CL in a Brazilian population. Moreover, two polymorphisms of the IL2RA gene were significantly and independently associated with CL. We confirmed this result in a second Brazilian sample (also exposed to L. braziliensis) and in Iranians infected with Leishmania tropica: IL2RA rs10905669 T (Pcombined = 6 × 10(-7)) and IL2RA rs706778 T (Pcombined = 2 × 10(-9)) were associated with greater susceptibility to lesion development. These alleles were also correlated with a poor IFN-γ response and poor FOXP3(+) regulatory T cell activation. Thus, IL-2 plays a crucial role in protection against the cutaneous ulcers caused by Leishmania, and the IL-2 pathway is a potential target for strategies aiming to control Leishmania-related diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia., Methods: From 56 clinical isolates we obtained a single amplified fragment (ca. 790 bp), which after cloning and sequencing resulted in 290 minicircle sequences from both active lesions and scars. We aimed to get a compositional profile of these sequences in clinical samples and evaluate the corresponding compositional changes. Sequences were analyzed with the compseq and wordcount (Emboss package) to get the composition of di-, tri-, tetra-, penta- and hexanucleotides. Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides., Results: This compositional analysis showed that minicircles amplified from active cutaneous lesions and scars have a distinct compositional profile as viewed by nucleotide composition of words up to 10mer. With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer., Conclusion: These results indicate that minicircle sequences can be monitored upon direct exposure to a selection/stressing environment (e.g. chemical action) by evaluating their nucleotide compositional profile. It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis is a neglected disease of humans in the New World that may also cause irreversible skin and eventually mucocutaneous lesions. This parasite can also infect dogs and represents a diagnostic challenge for veterinarians. Methods currently available for the diagnosis of ACL have a low sensitivity and may be time-consuming, representing a limit for treatment expedition of ACL. Quantitative real time PCR assays (qPCR) for the detection of L. (V.) braziliensis in canine blood samples were developed herein, and the detection limit and specificity of different molecular targets (kDNA and rDNA) evaluated. Of the protocols assessed, two qPCR assays, one targeting the kDNA and other the SSU rDNA of L. (V.) braziliensis, performed better, with detection limits of 100 fg and 10 pg, respectively. These assays were also used to test skin samples from humans with suspected ACL. The results indicate that the qPCR protocols developed represent an advance for the diagnosis of ACL in dogs and humans from this region, and provide a rapid and non-invasive diagnosis of the infection by L. (V.) braziliensis. Considering the quantitative nature of the assays, they will also be useful for monitoring treatment efficacy and preventing relapses in human patients in Brazil, although further studies are needed to critically evaluate the specificity of the qPCRs for their capacity to distinguish different Leishmania species and subspecies (represented by zymodemes) in other countries. Finally, molecular assays established may represent new tools for future basic and applied research focused on species identification, host-parasite associations, and infection dynamics in host and vector populations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

Studies suggest the influence of immune response on the successful treatment of American tegumentary leishmaniasis (ATL), and indicate the existence of protective immunity in self-healed patients. Thus, the aim of this work was to quantify interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL-) 10, IL-17, IL-22 and nitric oxide (NO) in culture supernatants of PBMC from patients with active disease (AD), after treatment (AT), and from self-healed (SH) and healthy subjects (CT), in response to Leishmania (Viannia) braziliensis insoluble antigen (AgIns). All groups of patients produced IFN-γ, indicating a predominant proinflammatory profile. AD and AT patients presented TNF-α levels, with a slight increase after therapy, whereas it was weakly quantified in SH. Interestingly, NO secretion was significant in these individuals, whereas IL-17 appeared in low levels and seems to be regulated by NO. Although IL-22 was detected in AD, its role is still questionable. The presence of IL-10 in all groups of patients suggests that the cytokine plays distinct roles in the disease. These results indicate that specific cellular immunity takes part against Leishmania, but with some similarities between the different clinical states herein described; these mediators seem to be necessary for the cure to occur., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve)=0.8 (95% CI=0.64-0.89) and AUC=0.90 (95% CI=0.75-0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

This study aims to investigate a flow cytometry performance-based methodology to detect anti-live (FC-ALPA-IgG) and anti-fixed (FC-AFPA-IgG) Leishmania (Viannia) braziliensis promastigote IgG as a means to monitor post-therapeutic cure of patients with localized cutaneous leishmaniasis (LCL). Serum samples from 30 LCL patients infected with L. (V.) braziliensis were assayed, comparing the IgG reactivity before and after specific treatment with pentavalent antimonial. Reactivities were reported as the percentage of positive fluorescent parasites (PPFP), using a PPFP of 60% as a cut-off value. In the serum dilution of 1:1024, the positive percentage of LCL serum sample for FC-ALPA-IgG and FC-AFPA-IgG was 86% and 90%, respectively, before treatment. Analysis of ∆PPFP that represents the difference between PPFP after and before treatment appeared as a new approach to monitor post-therapeutic IgG reactivity in LCL. Our data support the perspective of using FC-ALPA and FC-AFPA as a useful serologic tool for diagnosis and for post-therapeutic follow-up of LCL patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

American cutaneous leishmaniasis (ACL) has different clinical manifestations and these manifestations are dependent on the immunological status of the host. As CD4(+) and CD8(+) T cells and their mediators play a fundamental role in the host response to Leishmania and there is also a search for antigenic molecules to be used as future vaccines and tools for prognostic tests, this study characterized ACL patients' immune response after stimulation with soluble and insoluble fractions of L. (V.) braziliensis. We demonstrated a prevailing production of the Th2 cytokines, IL-4 and IL-10 and a specific production of IFN-γ and TNF-α in patients before treatment. There was also a predominance of CD4(+) T cells and a small percentage CD8(+) T cells. The insoluble antigenic fraction primarily stimulated CD4(+) T cells, while the soluble antigenic fraction showed a mixed profile, with CD4(+) T cells being the main responsible for Th2 cytokines and CD8(+) T cells for Th1 cytokines. Therefore, our results showed that a down-modulation of the Th1 type of response occurs in the initial phase of L. braziliensis disease, being the antigenic fractions capable of stimulating a specific immune response., (© 2011 Blackwell Publishing Ltd.)

WE REPORT TWO OCCUPATIONALLY ACQUIRED CASES OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL): one accidental laboratory autoinoculation by contaminated needlestick while handling an ACL lesion sample, and one acquired during field studies on bird biology. Polymerase chain reaction (PCR) assays of patient lesions were positive for Leishmania, subgenus Viannia. One isolate was obtained by culture (from patient 2 biopsy samples) and characterized as Leishmania (Viannia) naiffi through an indirect immunofluorescence assay (IFA) with species-specific monoclonal antibodies (mAbs) and by multilocus enzyme electrophoresis (MLEE). Patients were successfully treated with N-methyl-glucamine. These two cases highlight the potential risks of laboratory and field work and the need to comply with strict biosafety procedures in daily routines. The swab collection method, coupled with PCR detection, has greatly improved ACL laboratory diagnosis.

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R(2)=0.93, slope=-3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.

The conventional methods for identification and typing of Leishmania species depend on previous culture isolation of the parasites. Not infrequently, culture is unsuccessful and may result in misrepresentation of the heterogeneity of the original isolate. Thus, more reliable and precise identification of genotypes of Leishmania spp. is important for a better clinical and epidemiological understanding of the disease. We evaluated the potential of LSSP-PCR targeting kDNA minicircles in discriminating different variants of the parasite with the use of clinical samples directly or cultivated parasites. The 1st step of this procedure consists of the amplification of the minicircles by conventional PCR; the 2nd step is low-stringency amplification of the minicircles previously amplified, with the use of 1 of the primers. Although LSSP-PCR produced complex and distinct kDNA signatures for isolates representing different species, further experiments demonstrated that the approach had the potential for discriminating intraspecific variants of L. braziliensis. Thus, the generated profiles were too variable to be useful as markers for species identification. Moreover, we demonstrated that the approach can be directly applied to clinical samples. In conclusion, LSSP-PCR targeting kDNA minicircles produces profiles that reflect polymorphisms of the predominant classes of minicircles, and can be useful for studies aimed at discriminating Leishmania braziliensis genotypes without the need for previous cultivation of the parasite.

A cross-sectional seroepidemiological survey on canine leishmaniasis among pet dogs was carried out in an urban area in the State of Pernambuco (Brazil) where human cases of visceral leishmaniasis have sporadically been reported. Using an indirect fluorescent antibody test, anti-Leishmania antibodies were detected in 130 out of 322 dogs, confirming previous exposure to Leishmania parasites. The overall seroprevalence found was 40.3% (95% confidence interval: 34.9-45.9). Data analysis revealed that serological positivity was statistically associated with male (chi2=20.60, P-value=0.000) and juvenile dogs (chi2=4.24, P-value=0.039). Furthermore, it was observed that 85.3% of all seropositive dogs showed no clinical signs of leishmaniasis. The results showed a high seroprevalence of anti-Leishmania antibodies among dogs from an urban area of Pernambuco - with a large proportion of asymptomatic seropositive dogs - indicating that the prevalence of Leishmania infection in this area has been underestimated.

The aim of this study was to characterize the sandfly fauna in a military training area situated in the "Zona da Mata" region of Pernambuco State, Brazil, where human cases of American tegumentary leishmaniasis (ATL) had been observed, caused by Leishmania (Viannia) braziliensis. The occurrence of 16 sandfly species in the Marshall Newton Cavalcanti Military Training Camp (CIMNC) indicates an important diversity of sandfly species in this study area. Lutzomyia complexa was the most common species (51.36%) followed by L. choti (43.54%). All the captures were made in areas where training activities were conducted and where clinical cases had been detected in individuals performing training, following this period. The data relating the sandfly behavior to previous data in the same area provides strong evidence for the occurrence of an enzootic cycle of the parasite, and outbreaks followed the period of training activities in the remaining Atlantic Rain Forest and secondary forest areas.

Background: It is uncertain whether Leishmania parasites ever disappear after clinical cure of American cutaneous leishmaniasis (ACL). Recently, sensitive molecular techniques have allowed the identification of Leishmania parasites directly in specimens from patients' scars., Methods: Scars of 32 patients from northeastern Brazil who were treated and clinically cured of ACL were analyzed by use of polymerase chain reaction (PCR), culture, and histopathologic examination., Results: DNA specific for Leishmania (Viannia) was detected in scars of 30 (93.7%) of 32 patients. In specimens from 3 of the scars, Leishmania parasites could be isolated by culture; PCR results also were positive for those 3 specimens. No parasites were found by histopathologic examination, and fibrotic alterations were present in all cases, with slight inflammatory foci observed in 4 of the cases studied., Conclusions: The results suggest that clinical cure of ACL is rarely associated with sterile cure. The implications of persistence of parasites for the clinical evolution, relapse, and transmission of leishmaniasis deserves further studies, particularly with the increasing incidence of coinfection with leishmaniasis and acquired immunodeficiency syndrome.

Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations of Leishmania (Viannia) braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity of L. (V.) braziliensis strains from different regions where they are endemic. The results reinforce the clonal theory for Leishmania parasites showing the genetic diversity of this pathogen and an association of L. (V.) braziliensis genotypes with specific transmission cycles, probably reflecting an adaptation of different clones to the vector species involved.

PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.

An outbreak of American cutaneous leishmaniasis has been occurred in military training unit localized in 'Zona da Mata' of Pernambuco State, Brazil, where were registered 26 human cases. An epidemiological survey was carried out by entomological investigation and Montenegro skin test (MST). Lutzomyia choti presents predominance (89.9%) in sandflies identified. Out of 545 men who realized training activities were 24.12% positive to MST.

American cutaneous leishmaniasis (ACL) has been increasing in Pernambuco, thus becoming an important problem for Public Health. The incindence is predominant in the region called 'Zona da Mata', in the east of this state. This region corresponds geographically to the primitive area of the Atlantic forest. In order to characterize the eco-epidemiology expression of ACL in this region, two localities situated in the municipalities of Amaraji e Cortes have been selected by the criterion of higher incindence of human cases. Five stocks of patients were characterized and identified on the basis of enzyme profiles as a new variant of Leishmania (V.) braziliensis. A survey of wild and domestic animals was carried out by means of a parasitological and serological diagnosis. Through the analysis of the spleen and liver imprints, were detected amastigotes compatible with Leishmania in five Nectomys s. squamipes, five Bolomys 1. pixuna, two Rattus r. alexandrinus and one Rattus r. frugivorus. For two years we carried out monthly sandflies captures using CDC light traps as well as manual captures. Lutzomyia whitmani was predominant, which accounted for 97.4% of the total. These data indicate a strong evidence on the vector and the potential reservoirs of L. braziliensis in this region.