Increased Risk of American Tegumentary Leishmaniasis in an Urban and Rural Area of Caratinga, Brazil between 2016 and 2021.

We used spatial analysis tools to examine the epidemiological situation and spatial distribution of American tegumentary leishmaniasis in the municipality of Caratinga between 2016 and 2021. In addition, potential sandfly vectors were captured. All information used in this study was retrieved from public health archives and confirmed in the state health services databases. All cases were analyzed using Geographic Information Systems software. In addition, sandfly collections and molecular detection of Leishmania were carried out in areas with the highest number of cases. During the analyzed period, American tegumentary leishmaniasis (ATL) cases increased and remained high in the last years. The hotspots included urban areas of Caratinga city and the districts of Patrocínio of Caratinga and Sapucaia. The species Nyssomyia whitmani, Nyssomyia intermedia, and Migonemyia migonei were the most abundant species and the ITS1-polymerase chain reaction technique detected Leishmania DNA in these species. On the basis of our analyses, the urbanization of ATL in Caratinga has taken place in recent years. Because of the increase in the number of human cases and the presence of vectors, it is recommended that health authorities focus on control measures in hotspots.

Characterization of Differentially Abundant Proteins Among Leishmania (Viannia) braziliensis Strains Isolated From Atypical or Typical Lesions.

Leishmania (Viannia) braziliensis is the main etiological agent of cutaneous and mucocutaneous leishmaniasis in Latin America. Non-ulcerated atypical tegumentary leishmaniasis cases caused by L. braziliensis have been reported in several regions of the American continent, including the Xacriabá indigenous reserve in São João das Missões/Minas Gerais, Brazil. Parasites isolated from these atypical clinical lesions are resistant to antimony-based therapeutics. In the present study, proteins displaying differential abundance in two strains of L. braziliensis isolated from patients with atypical lesions compared with four strains isolated from patients with typical lesions were identified using a quantitative proteomics approach based on tandem mass tag labeling (TMT) and mass spectrometry. A total of 532 (P<0.05) differentially abundant proteins were identified (298 upregulated and 234 downregulated) in strains from atypical lesions compared to strains from typical lesions. Prominent positively regulated proteins in atypical strains included those that may confer greater survival inside macrophages, proteins related to antimony resistance, and proteins associated with higher peroxidase activity. Additionally, we identified proteins showing potential as new drug and vaccine targets. Our findings contribute to the characterization of these intriguing L. braziliensis strains and provide a novel perspective on Atypical Cutaneous Leishmaniasis (ACL) cases that have been associated with therapeutic failures.

A follow-up study (2007-2018) on American Tegumentary Leishmaniasis in the municipality of Caratinga, Minas Gerais State, Brazil: Spatial analyses and sand fly collection.

BACKGROUND: The municipality of Caratinga is an important endemic area for American Tegumentary Leishmaniasis (ATL) and no epidemiological studies were performed during the past two decades. Here, we analyzed the epidemiological situation and the geographical distribution of ATL cases in the municipality of Caratinga from 2007 to 2018 using geographic information systems (GIS). Also, we evaluated the impact of several demographic parameters in ATL distribution and the sand flies incriminated in its transmission. METHODS: All demographic information (gender, age, educational level, clinical form, diagnostic criteria and case evolution) used in this study was retrieved from the public health archives and confirmed in the State Health Services databases. All cases were analyzed using GIS software based on ATL distribution. Also, non-systematic sand fly collections and molecular detection of Leishmania were performed in the hotspots. RESULTS AND CONCLUSIONS: During the period, ATL cases continued and increased especially in the past years (2016-2018). Hotspots included urban Caratinga areas and the districts of Patrocínio de Caratinga and Sapucaia. The species Nyssomyia whitmani, Nyssomyia intermedia, Migonemyia migonei and Evandromyia cortelezzii complex were captured. However, ITS1-PCR did not detect Leishmania DNA in those insects. Based on our analyses, urbanization of ATL in Caratinga has occurred in the past years. Due to the increase in the number of cases and vectors presence, it is recommended that health authorities focus on control measures in the most affected areas (Patrocínio of Caratinga and Sapucaia districts and urban Caratinga).

Visceral leishmaniasis: a practical strategy for quantitative molecular diagnosis in naturally infected dogs.

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.

Leishmania presence in bats in areas endemic for leishmaniasis in central-west Brazil.

Leishmaniasis involves the participation of several species of both wild and domestic mammal hosts and sandfly vectors, which demonstrates the eco-epidemiological complexity observed in this disease. Bats are among the most abundant types of mammals and the scarcity of research on Leishmania infection in these animals gives evidence of the importance of new studies that aim to clarify this relationship. This study aimed to detect the Leishmania spp. in bats. 146 bats, representing 16 different species belonging to the Molossidae, Vespertilionidae, and Phyllostomidae families, were received and processed for collection of tissues. Skin samples were collected from 100% of the bats, and liver samples were collected from 87% (n = 127). After evaluating the quality of the DNA extracted by means of PCR directed to the IRBP gene, the samples considered suitable for the Leishmania detection test were submitted for PCR directed to Leishmania kDNA, and to confirm positivity, were tested to the SSUrRNA gene-directed Nested-PCR. The Leishmania presence in the species Molossus pretiosus, Nyctinomops macrotis, and Lasiurus cinereus are the first reports this encounter in these species of bats in Brazil. Furthermore, new species of bats as possible hosts for L. infantum are reported, such as Molossus pretiosus, Myotis nigricans, Nyctinomops laticaudatus, Nyctinomops macrotis, and, for L. braziliensis, Lasiurus cinereus and Cynomops planirostris. These findings in bats in an area endemic for leishmaniasis indicate that these animals may be involved in sustaining the disease cycle in this location.

Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs.

The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that amplify a 300-350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4 (4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania amazonensis, and their serum samples tested positive by IFAT and DPP. These findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the first record of Leishmania (Leishmania) amazonensis confirmed by a specific molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.

TipMT: Identification of PCR-based taxon-specific markers.

BACKGROUND: Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. RESULTS: Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. CONCLUSIONS: The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil.

The double stranded RNA (dsRNA) virus Leishmaniavirus (Totiviridae) was first described in Leishmania guyanensis and L. braziliensis (LRV1), and more recently from L. major and L. aethiopica (LRV2). Parasites bearing LRV1 elicit a higher pro-inflammatory profile, arising through activation of Toll like receptor 3(TLR3) interacting with the viral dsRNA. LRV1 is most common in Leishmania from the Amazon region; however data for other regions of Brazil are more limited. Here we applied PCR tests with validated 'universal' LRV1 primers to search for LRV1 in 40 strains of cultured L. braziliensis from several locales within Minas Gerais State, including patients presenting with atypical lesion pathology. All strains were negative however. These data are in agreement with results from other areas of Southeastern Brazil that LRV1 is relatively uncommon.

Mitochondrial genetic variability of Didelphis albiventris (Didelphimorphia, Didelphidae) in Brazilian localities.

Didelphis albiventris is a well-known and common marsupial. Due to its high adaptability, this very widespread generalist species occurs under various environmental conditions, this even including protected regions and disturbed urban areas. We studied a 653 bp fragment of cytochrome oxidase c (COI) from 93 biological samples from seven Brazilian localities, with linear distances ranging between 58 and about 1800 km to analyze the effects of geographic distances on variability and genetic differentiation. The haplotype network presented nine haplotypes and two genetic clusters compatible with the two most distant geographic areas of the states of Minas Gerais, in the southeast, and Rio Grande do Sul, in the extreme south. As each cluster was characterized by low nucleotide and high haplotype diversities, their populations were obviously composed of closely related haplotypes. Surprisingly, moderate to high F(ST) differentiation values and a very weak phylogeographic signal characterizes interpopulation comparisons within Minas Gerais interdemes, these being correlated with the presence of privative haplotypes. On a large rgeographic scale, a comparison between demes from Minas Gerais and Rio Grande do Sul presented high F(ST) values and a robust phylogeographic pattern. This unexpected scenario implies that mtDNA gene flow was insufficient to maintain population cohesion, reflected by the observed high differentiation.

Wild, synanthropic and domestic hosts of Leishmania in an endemic area of cutaneous leishmaniasis in Minas Gerais State, Brazil.

Domestic, synanthropic and wild hosts of Leishmania spp. parasites were studied in an area endemic for American tegumentary leishmaniasis (ATL), specifically in northern Minas Gerais State, Brazil. Domestic dogs and small forest mammals are reservoir hosts for L. (Leishmania) infantum. However, the role that these animals play in the transmission cycle of the Leishmania spp. that cause cutaneous leishmaniasis is not well known. This study evaluated 72 rodents, 25 marsupials and 98 domestic dogs found in two villages of the Xakriabá Indigenous Territory, an area of intense ATL transmission. A total of 23 dogs (23.47%) were shown to be positive according to at least one test; 8 dogs (8.16%) tested positive in a single serological test and 15 dogs (15.31%) tested positive by IFAT and ELISA. Eleven dogs were euthanised to allow for molecular diagnosis, of which nine (81.8%) tested positive by PCR for Leishmania in at least one tissue. Seven animals were infected only with L. (L.) infantum, whilst two displayed a mixed infection of L. (L.) infantum and L. (V.) braziliensis. Isoenzymatic characterisation identified L. (L.) infantum parasites isolated from the bone marrow of two dogs. Of the 97 small mammals captured, 24 tested positive for Leishmania by PCR. The results showed that L. (V.) braziliensis, L. (L.) infantum and L. (V.) guyanensis are circulating among wild and synanthropic mammals present in the Xakriabá Reserve, highlighting the epidemiological diversity of ATL in this region.

Molecular detection of Leishmania braziliensis in Rattus norvegicus in an area endemic for cutaneous leishmaniasis in Brazil.

Leishmania nested PCR (LnPCR) targeted to the SSUrRNA gene and DNA sequencing were used to analyze 315 tissue samples from 80 Rattus norvegicus specimens trapped in an area endemic for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil. Of the samples analyzed, 17.46% (55/315) of all tissues, 10% (8/80) of skin, 26.92% (21/78) of blood, 30.76% (24/78) of bone marrow and 2.53% (2/79) of spleen were positive for Leishmania. The overall infection prevalence was 36.25% (29/80) The DNA sequencing showed that 65.51% (19/29) of the positive animals were infected by parasites belonging to the Leishmania braziliensis complex. The identification of L. braziliensis DNA in R. norvegicus in an area with a high prevalence of leishmaniasis might imply a zoonotic role of this species. The rodent control programs and health education may represent important measures toward the control of leishmaniasis.

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom.

BACKGROUND: Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms. METHODS: The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity. RESULTS: Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H2O2. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC(50) of 0.07µM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase. CONCLUSION: Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells. GENERAL SIGNIFICANCE: These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis.

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Study of phlebotomine sand flies (Diptera: Psychodidae) collected in a Leishmania-endemic area of the metropolitan region of Belo Horizonte, Brazil.

Phlebotomine sand flies are distributed across nearly all faunal regions of the world, represented by over 800 species, of which many are important vectors of human pathogens. Brazil is currently faced with the expansion and urbanization of leishmaniases, with an increase in the numbers of human cases and seropositive dogs in various medium-sized to large cities. The objective of the current study was to survey the phlebotomine sand fly species in an area endemic for American cutaneous leishmaniasis (ACL) and American visceral leishmaniasis (AVL), i.e., the municipal district of Santa Luzia, lying within the metropolitan region of Belo Horizonte in the Brazilian State of Minas Gerais. Sand flies were collected monthly in 2004-2005 using modified Falcão light traps hung in the peridomiciles of houses and surrounding wooded areas in the district of Baronesa. A total of 1,552 sand flies belonging to seven species was collected, and an interesting pattern of the distribution of the most abundant species relative to the sampling locality was revealed. In the wooded areas Lutzomyia whitmani (Antunes & Coutinho) predominated, whereas in the urban area Lutzomyia longipalpis (Lutz & Neiva) was the most abundant species. These results indicate two possible epidemiological patterns of Leishmania transmission in Santa Luzia: one for American cutaneous leishmaniasis associated predominantly with wooded areas, and another for AVL, with transmission principally occurring around human habitations.

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100 percent, 55 percent and 22 percent of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (= Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (= Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil.

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.

Didelphis marsupialis (common opossum): a potential reservoir host for zoonotic leishmaniasis in the metropolitan region of Belo Horizonte (Minas Gerais, Brazil).

Identification of the zoonotic reservoir is important for leishmaniasis control program. A number of (wild) animal species may serve as reservoir hosts, including the opossum Didelphis marsupialis. A survey carried out in Didelphis specimens (n = 111) from the metropolitan region of Belo Horizonte, an important focus of human leishmaniasis in Brazil, is reported. All animals were serologically tested with indirect fluorescence antibody test (IFAT) and direct agglutination tests (DAT) based on L. (L.) donovani or L. (V.) braziliensis antigen. A sub-population (n = 20) was analyzed with polymerase chain reaction (PCR) for the presence of Leishmania-specific DNA. For species identification, PCR-positive samples were subjected to restriction enzyme fragment polymorphism (RFLP) analysis. Depending on the sero-diagnostic test employed, the sero-prevalence varied between 8.1% (9/111 animals positive with DAT test based on L. braziliensis antigen) and 21.6% (24/111 animals positive with IFAT). Five out of 20 samples analyzed with PCR tested positive for the presence of Leishmania-specific DNA. RFLP analysis revealed that two samples contained L. braziliensis complex DNA, one contained L. donovani complex DNA, and two samples could not be typed with the methodology used. These data suggest a potential role for the opossum as a reservoir host for zoonotic leishmaniasis in the region.

Contribution of molecular techniques to the epidemiology of neotropical Leishmania species.

Brandão-Filho et al. and Oliveira et al. have recently reported the detection of Leishmania in sylvatic and synanthropic animals that were captured in areas of Brazil that are endemic for leishmaniasis. Such investigations raise the issue of reservoirs of important endemic diseases by using modern molecular biology and biochemical techniques that complement traditional methods. Ecoepidemiological studies focusing on possible reservoirs have been important for providing contributions to prophylaxis and control measures to be employed by public health authorities.