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Genus and species of phlebotomine sand flies have been recorded and described in caves in Brazil, but no study has provided the food source used by sand flies in these environments. Herein, we identified the blood source used by sand fly species in caves located at “Quadrilatero Ferrifero” (QF), Minas Gerais state. Specimens were manually collected near or on anurans inside ferruginous caves in Serra do Gandarela National Park and Serra do Rola Moca State Park. Males and females were placed in vials with 70% alcohol and stored at −10°C. Females engorged, after specific identification, had DNA extracted and followed for PCR amplification using specific primers. Sequencing was analyzed in the GenBank and Barcode of Life. A total of 198 specimens were collected (107 females and 91 males), all of them belonging to species Sciopemyia aff. microps (88.89%), Sciopemyia sordellii (10.61%), or Martinsmyia oliveirai (0.50%). When it comes to the females, 89 were S. aff. microps and 18 S. sordellii. Nineteen engorged females of S. aff. microps were analyzed and most of them (n=18) presented blood from Bokermannohyla martinsi and one contained blood from Scinax fuscovarius. The blood present in engorged females of S. sordellii (n=4) was from B. martinsi. Sciopemyia genus specimens are commonly found in collections carried out inside natural caves, but this was the first study to prove that females of this genus feed on cold-blooded animals in nature. • Here we proved that sand flies feed in cold-blooded animals in in Brazilian caves. • Females of the Sciopemyia genus were for the first time found feeding in natural habitats. • Anurans of the family Hylidae were identified as source by molecular analyzes. • Insect bloodmeal identification can help assessing the fauna in several biomes. • This is the first record of S. aff. microps in caves of Brazil.

The poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund’s Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE®) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.

The understanding on the role of bats in the ecology of zoonotic diseases, especially its relevance as a carrier of pathogens, is important for the determination of preventive measures considering the One Health context. The present study aimed to investigate the presence of Brucella spp., Leptospira spp. and Salmonella spp. in blood (n = 163), liver (n = 35) and spleen (n = 62) samples from bats captured in Montes Claros, Minas Gerais, Brazil. Only Salmonella spp. was found in a blood sample of an insectivorous female bat of the species Lasiurus blossevilli, evidencing the capacity of this animal species to host this pathogen. In conclusion, our results in bats from Montes Claros indicate that they do not act as hosts for Brucella spp. and Leptospira spp., although being potential carriers of Salmonella spp. in a low prevalence.

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.

Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named “L. major-like”, and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major-like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major-like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World.

This study aimed to determine the occurrence of Leishmania infection in bats in urban and wild areas in an endemic municipality for visceral and cutaneous leishmaniasis in Minas Gerais, Brazil. Between April 2014 to April 2015, 247 bats were captured and classified into 26 species belonging to Phyllostomidae (90.7%), Vespertilionidae (8.1%) and Molossidae (1.2%) families. Blood samples from 247 bats were collected and submitted to nested-PCR, targeting the variable V7-V8 region of the SSU rRNA gene, followed by sequencing of the PCR product. The overall infection rate of Leishmania spp. in bats was 4.4%. Of the eleven bats infected, ten were frugivorous bats: Artibeus planirostris (8/11), Artibeus lituratus (1/11) and Artibeus cinereus (1/11) and one a nectarivorous bat (Glossophaga soricina). None of the individuals exhibited macroscopic alterations in the skin, spleen or liver. Phylogenetic analysis separated Leishmania species in clades corresponding to the subgenera Viannia, Leishmania, and Mundinia, and supported that the isolates characterized in the present study clustered closely with Leishmania (Viannia) sp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis. Here we report for the first time the bat Artibeus cinereus as a host of Leishmania (Leishmania) amazonensis. In the study we found that the mean abundance of bats did not differ in wild habitats and urban areas and that bat-parasite interactions were similarly distributed in the two environments. On the other hand, further studies should be conducted in more recent times to verify whether there have been changes in these parameters.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

SUMMARYIn South America, visceral leishmaniasis is frequently caused byLeishmania infantumand, at an unknown frequency, byLeishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections withL. amazonensisandL. infantumin a hamster model. We show that mixed infections are associated with more severe clinical disease than infection withL. amazonensisorL. infantumalone. In spleens with mixed infections,L. infantumoutcompetedL. amazonensisin the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected withL. infantum.Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected withL. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected withL. infantum. This finding has important implications regarding the biology ofLeishmaniaand humoral immune responses to infections with these organisms.

Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Parasitologia. Belo Horizonte, MG, Brazil. Universidade Federal de Goi?s. Instituto de Patologia Tropical e Sa?de P?blica. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Instituto de Patologia Tropical e Sa?de P?blica. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal de Uberl?ndia. Instituto de Ci?ncias Biom?dicas. Departamento de Imunologia, Microbiologia e Parasitologia. Minas Gerais, MG, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Belo Horizonte, MG, Brazil Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. 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.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

© 2015 Elsevier Inc. The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the BALB/c mice proved to be similar when comparing the F2 and F4 fractions with a known Th1 adjuvant (saponin), though animals vaccinated with saponin did present a slight to moderate inflammatory edema on their hind footpads. In conclusion, the F2 and F4 fractions appear to induce a Th1-type immune response and, in this context, they could be evaluated in association with other protective antigens against Leishmania, as well as in other disease models., Instituto Nacional de Ciencia e Tecnologia em Nano-biofarmaceutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8 and APQ-488237/2013-0).

Leishmaniasis is a neglected tropical disease caused by >20 species of the protozoan parasite Leishmania . Meglumine antimoniate (Glucantime) is the first-choice drug recommended by the World Health Organization for the treatment of all types of leishmaniasis. However, the mechanisms of action and toxicity of pentavalent antimonials, including genotoxic effects, remain unclear. Therefore, the mechanism by which meglumine antimoniate causes DNA damage was investigated for BALB/c mice infected by Leishmania ( Leishmania ) infantum and treated with meglumine antimoniate (20 mg/kg for 20 days). DNA damage was analyzed by a comet assay using mouse leukocytes. Furthermore, comet assays were followed by treatment with formamidopyrimidine-DNA glycosylase and endonuclease III, which remove oxidized DNA bases. In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the animals' sera were assessed. To investigate mutagenicity, we carried out a micronucleus test. Our data demonstrate that meglumine antimoniate, as well as L. infantum infection, induces DNA damage in mammalian cells by the oxidation of nitrogenous bases. Additionally, the antileishmanial increased the frequency of micronucleated cells, confirming its mutagenic potential. According to our data, both meglumine antimoniate treatment and L. infantum infection promote oxidative stress-derived DNA damage, which promotes overactivation of the SOD-CAT axis, whereas the SOD-GPx axis is inhibited as a probable consequence of glutathione (GSH) depletion. Finally, our data enable us to suggest that a meglumine antimoniate regimen, as recommended by the World Health Organization, would compromise GPx activity, leading to the saturation of antioxidant defense systems that use thiol groups, and might be harmful to patients under treatment.

The aims of the study were to determine the blood feeding preferences of sandflies and to identify species of Leishmania that infected phlebotomines in Caxias, Maranhão, Brazil, an area that is highly endemic for leishmaniasis. Sandflies were captured in light traps located in the peridomiciliary environments of randomly selected houses in urban and rural settings between 1800 and 0600 hours on new moon days between March 2013 and February 2015. DNA extracts from 982 engorged female sandflies were submitted to fragment length polymorphism analysis to identify infecting species of Leishmania, and blood sources were identified for 778 of these specimens. Infection by Leishmania infantum was detected in Lutzomyia longipalpis, Lu. whitmani and Lu. termitophila; L. infantum/L. braziliensis in Lu. longipalpis, Lu. whitmani and Lu. trinidadensis; L. shawi in Lu. longipalpis; L. mexicana in Lu. longipalpis; L. braziliensis in Lu. longipalpis and Lu. whitmani; L. guyanensis in Lu. longipalpis and Lu. termitophila; L. amazonensis in Lu. longipalpis and L. lainsoni or L. naiffi in Lu. longipalpis, while Lu. longipalpis and Lu. trinidadensis were infected with unidentified Leishmania sp. Blood sources were identified in 573 individual phlebotomines and the preferred hosts were, in decreasing order, chicken, dog, rodent and human with lower preferences for pig, horse, opossum and cattle. Lu. longipalpis and Lu. whitmani performed mixed feeding on man, dog and rodent, while Lu. longipalpis was the most opportunistic species, feeding on the blood of all hosts surveyed, but preferably on dog/chicken, dog/rodent and rodent/chicken. Our findings reveal the concomitant circulation of Leishmania species that cause visceral leishmaniasis and tegumentary leishmaniasis in the study area, and explain the occurrence of autochthonous human cases of both clinical forms of leishmaniasis in Caxias, Maranhão. The results support our hypothesis that, in the municipality of Caxias, transmission of Leishmania occurs in close proximity to humans.

Species of the genus Spirometra are diphyllobothriid tapeworms with complex life cycles and are involved in human sparganosis, a neglected disease that affects individuals worldwide. Although some species were reported in wild felids and human cases of sparganosis were described in Brazil, the biology and taxonomy of these parasites are poorly understood. In the present study, samples of diphyllobothriids (eggs and/or proglottids) obtained from the stools of wild carnivores (Leopardus pardalis and Lycalopex vetulus) and plerocercoid larvae found in a snake (Crotalus durissus) from Brazil were analysed by amplifying a fragment of the gene cytochrome c oxidase subunit 1 (cox 1). The DNA sequences obtained here for the first time from the Spirometra spp. from Brazil were used to evaluate the phylogenetic relationships with other species. Molecular data identified two species in the Brazilian samples (evolutionary divergence of 17.8-19.2%). The species were identified as Spirometra sp. 1, found in Le. pardalis, and Spirometra sp. 2 found in Ly. vetulus and C. durissus, and they differed from Asian isolates of Spirometra erinaceieuropaei (17.5-20.2% and 12.2-15.6%, respectively), a species previously considered to be distributed worldwide. Moreover, Spirometra sp. 1 is genetically distinct from Sparganum proliferum from Venezuela (19.6-20.4%), while Spirometra sp. 2 is more closely related with the Venezuelan species (6.1-7.0%). Sequences of Spirometra sp. 2 revealed that it is conspecific with the Argentinean isolate of Spirometra found in Lycalopex gymnocercus (1.9-2.2%). Taxonomic and phylogenetic aspects related to New World species of Spirometra are briefly discussed.

The potential of Gafanhoto Park as an American cutaneous leishmaniasis (ACL) focus was evaluated by examination of sand fly vectors of the Leishmania parasite. This forest remnant is located in a periurban area of Divinopolis, Brazil, where autochthonous cases of ACL have been reported. Sand fly populations were monitored over a 2-yr period (2006–2008) by using light traps (HP and Shannon). During systematic collections with HP traps, 824 specimens in total (342 males and 482 females) of 21 species were captured. Most prevalent species were as follows: Brumptomyia brumpti (Larrouse), Lutzomyia aragaoi (Costa Lima), Lutzomyia lutziana (Costa Lima), Lutzomyia sordellii (Shannon & Del Ponte), and Lutzomyia whitmani (Antunes & Coutinho). Using Shannon traps, 257 specimens representing 15 species were collected (159 females and 98 males), with a high prevalence of L. whitmani and Lutzomyia neivai (Pinto), both vectors of Leishmania braziliensis (Vianna). To ascertain the level of natural infection, a sample of females captured in Shannon traps was assayed for the presence of Leishmania by using polymerase chain reaction-restriction fragment length polymorphism, where 39% of insects were positive. The most infected species was L. whitmani (29 sand flies; 18.2%), followed by L. neivai (21; 13.2%), Lutzomyia christenseni (Young & Duncan) (five; 3.1%), Lutzomyia pessoai (Coutinho & Barreto) (three; 1.9%), L. aragaoi (one; 0.6%), Lutzomyia fischeri (Pinto) (one; 0.6%), Lutzomyia lenti (Mangabeira) (one; 0.6%), L. lutziana (one; 0.6%), and Lutzomyia monticula (Costa Lima) (one; 0.6%). The finding of potential and incriminated vectors naturally infected with Leishmania reinforces the need of epidemiologic surveillance in the area.

The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.

The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

Background The Lençóis Maranhenses National Park, located in Maranhão, Brazil, is a region of exceptional beauty and a popular tourist destination. The adjoining area has suffered from the impact of human activity and, consequently, has experienced outbreaks of leishmaniasis. This study aimed to evaluate the composition, abundance, species richness and seasonal distribution of sand flies in the region and to determine the constancy of the insect population. Methods The survey was conducted at three sites located in the municipalities of Barreirinhas and Santo Amaro between September 2012 and August 2013. Sampling was performed monthly using automatic light traps installed 1.5 m above the soil adjacent to 13 randomly selected rural dwellings. At each site, one trap was placed in the peridomicile near to animal enclosures and another (extradomicile) at 500 m from the peridomicile. Results A total of 4,474 individual sand flies were collected over the year with the highest abundance recorded during the rainy season (December to June). Nine species were collected: L. whitmani, L. longipalpis, L. lenti, L. sordellii, L. evandroi, L. flaviscutellata, L. wellcomei, L. termitophila and L. intermedia. Although peridomiciliary and extradomiciliary environments presented similar species richness, the Shannon diversity index was significantly lower in the former (H’ = 2.4) compared with the latter (H’ = 4.98). Lutzomyia whitmani and L. longipalpis were the most abundant species and were classified as constant (constancy index, CI = 100 %) along with L. lenti (CI = 58.3), L. evandroi (CI = 58.3) and L. sordellii (CI = 66.7). The remaining four species presented CI values between 25 and 50 % and were considered accessory. Conclusions The present results confirm the present of L. whitmani and L. longipalpis in the peridomicile of houses in Lençóis National Park. The abundance of these species could explain, respectively, the endemicity of cutaneous leishmaniasis and sporadic cases of visceral leishmaniasis in the study area. However, in the case of cutaneous leishmaniasis, the presence of other sand fly vectors (in addition to L. whitmani) cannot be neglected. Finally, this study emphasizes the need for a more effective and permanent supervision to control the expansion of these vectors and leishmaniasis outbreaks.

The richness, relative abundance and the monthly frequency of species of sandflies (Diptera,Psychodidae) in an urban area of the municipality of Codo, Maranhao, a state that records a highprevalence of visceral leishmaniasis, was studied. The insects were captured with CDC light traps,from 18 h to 6 h, twice per month from June 2012 to May 2013 and 5,722 specimens of 11 species, 10belonging to the genus Lutzomyia and one to the genus Brumptomyia were collected. The abundancewas higher outdoors (4,009 samples: 66% males and 34% females) than in households (1,713 samples:55.9% males and 44.1% females). The most abundant species was L. longipalpis (97.7%), whichtogether with L. evandroi, L. sordellii and L. termitophila were more constant throughout the year. Thefrequency of sandflies was higher in the dry season (57.9%), rising with increasing mean temperature(p=0.0156, r=0.1007) and showed a decrease with increasing relative humidity (p=0.0001, r=-0.1740).Vector control during the dry season, when sandflies are most abundant, is suggested.

Background Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. Methods Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. Results LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF–α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). Conclusion The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

Hantaviruses are zoonotic viruses harbored by rodents, bats, and shrews. At present, only rodent-borne hantaviruses are associated with severe illness in humans. New species of hantaviruses have been recently identified in bats and shrews greatly expanding the potential reservoirs and ranges of these viruses. Brazil has one of the highest incidences of hantavirus cardiopulmonary syndrome in South America, hence it is critical to know what is the prevalence of hantaviruses in Brazil. Although much is known about rodent reservoirs, little is known regarding bats. We captured 270 bats from February 2012 to April 2014. Serum was screened for the presence of antibodies against a recombinant nucleoprotein (rN) of Araraquara virus (ARAQV). The prevalence of antibody to hantavirus was 9/53 with an overall seroprevalence of 17%. Previous studies have shown only insectivorous bats to harbor hantavirus; however, in our study, of the nine seropositive bats, five were frugivorous, one was carnivorous, and three were sanguivorous phyllostomid bats.

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.

In an attempt to isolate and characterize Toxoplasma gondii from the State of Minas Gerais, Brazil, musculature samples from 72 pigs, 25 dogs, 28 free-range chickens and 50 chickens produced in industrialized farms were collected. Antibodies to T. gondii have not been detected in pigs, but were found in nine (40.9 %) out of 22 dogs, and in 15 (53.6 %) of 28 free range chickens. T. gondii was not isolated from pigs and industrialized chickens, but from eight dogs and 11 free range chickens. In order to determine T. gondii virulence, female BALB/c mice were inoculated with 10(3), 10(2), 10(1) and 10(0) tachyzoites of the 19 isolates. The strains RH (virulent) and ME49 (non-virulent) were used as references. Isolates were divided into three groups according to the virulence phenotype: five isolates were classified into virulent in mice, one into non-virulent and 13 into intermediate virulent. Nested-PCR of T. gondii SAG2 locus amplified DNA from 21 out of 22 DNA samples directly extracted from heart of free range chickens. These samples were genotyped through a PCR-RFLP assay. Seventeen (80.9 %) were classified into type I; one (4.8 %) into type III and three (14.3 %) into type I or II.

Taxic responses may play a role in development of Leishmania in their phlebotomine sand fly vectors. They are possibly responsible for movement of the parasites towards the anterior regions of the gut, from where they would be transmitted to the vertebrate host. A methodology capable to distinguish chemotaxic from osmotaxic responses was described and used to characterise taxic responses in Leishmania promastigotes. These were able to respond to chemotaxic as well as to osmotaxic stimuli. Like bacteria, promastigotes were capable to undergo "adaptation," a phenomenon by which they stop responding to a continuos stimulus. A model capable to explain how a relatively small number of different receptors works to perceive gradients in chemotaxic responses was proposed. According to this model, these receptors possess low specificity and a wide range of affinities varying from high to low. A low specificity makes the same receptor able to bind to a large number of different but structurally related molecules and; a wide range of affinities (considering a population of receptors), implies that the number of receptors "occupied" by attractant molecules along a gradient would go growing step by step.

Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) at eight independent loci was used to determine the types (I, II or III) lineage of 20 Toxoplasma gondii strains isolated from humans and animals in Brazil. RH (type I, highly virulent), ME49 (type II, avirulent) and VEG (type III, avirulent) were used as reference strains. Differently from expected frequencies, all Brazilian strains showed to have recombinant genotypes, with typical alleles of types I, II or III at almost all loci assessed. The cB21-4 locus, a microsatellite marker, showed a higher allelic polymorphism with seven alleles among strains under analysis. Data have also shown that many Brazilian T. gondii strains presented a new haplotype at the L363 locus. When results of the eight loci were combined, 14 schizodemes were characterized out of the 20 T. gondii strains isolated in Brazil. The phenogram representing PCR–RFLP data separated Brazilian strains into two distinct genetic groups associated with murine virulence phenotype, termed groups I-A and I-B. Strains from group I-A (AS28, BV and N) that were highly virulent in BALB/c mice, were clustered with RH reference strain. Only those strains presented the haplotype I at the L363 locus, suggesting that this could be a possible marker of highly virulent strains. Strains from group I-B (cystogenic strains) showed a more heterogeneous behavior regarding virulence: a few of them (EGS, RAR, SAF, D5 and D6) were virulent, others (C4, P and D8) avirulent and most of them (D1, D2, D3, D4, D7, EFP, CH1, CH2 and CH3) intermediate virulent in mice. A significant linkage disequilibrium was observed in the population surveyed. However, the role of sexual recombination in the population structure of T. gondii in Brazil seems to be more central than in Europe and North America, where most studies have been performed.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

Genetic relationships between Eimeria parasites of domestic fowl, including three isolates of E. acervulina, one of E. maxima, four of E. mitis, six of E. praecox, one of E. tenella, and two of uncertain identity, were analyzed by random amplified polymorphic DNA (RAPD) and the unweighted pair-group method with arithmetic mean, using 12 primers. Each primer amplified 4-34 DNA fragments/isolate. The similarity coefficients and phenograms were calculated from RAPD products with 230-2,000 bp in silver-stained polyacrylamide gels. Some primers generated RAPD markers which were species-specific for E. acervulina, E. mitis, and E. praecox. The phenograms revealed six clusters, each corresponding to an individual Eimeria species. The pBP and pBP2 isolates, of uncertain identity, clustered with the E. acervulina isolates (100% bootstrap). The intraspecific relationships showed certain degree of genetic isolation of the Eimeria populations, and it was associated with broiler house and with geographic separation.

Screening for digestive glycosidases in different parts of the gut and associated organs of Lutzomyia longipalpis is reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an alpha-glucosidase, an N-acetyl-beta-d-glucosaminidase, an N-acetyl-beta-d-galactosaminidase, and an alpha-l-fucosidase. Their possible role and importance for Leishmania development are discussed and the alpha-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data and Leishmania development is proposed.

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic diseaseParâmetros de virulência na caracterização de Cepas de Entamoeba histolytica. A caracterização quanto a virulência das diferentes cepas de E. histolytica foi avaliada através de ensaios in vivo e in vitro. Discrepâncias nesta caracterização têm surgido quando se compara os diferentes ensaios. Avaliamos alguns parâmetros de virulência na caracterização de 5 cepas axênicas de E. histolytica. Estas cepas foram caracterizadas com relação à sua capacidade para induzir abscesso em fígado de hamster, eritrofagocitose e efeito citopático sobre células VERO. Encontramos correlação significante entre os ensaios biológicos in vitro, mas não entre estes com os ensaios in vivo. Boa correlação foi achada entre o efeito citopático e o número médio de eritrócitos ingeridos, mas não com a percentagem de amebas que fagocitaram eritrócitos, mostrando que grande variabilidade pode ser observada no mesmo ensaio, dependendo da variável escolhida. Não foi possível correlacionar isoenzima e padrão de fragmento de restrição com o índice de virulência desde que todas as cepas estudadas apresentaram padrão patogênico. Os resultados discordantes observados nos diferentes ensaios de virulência sugerem que a mesma não deve ser avaliada diretamente. O que de fato está sendo avaliado são as diferentes características biológicas ou funções do parasita, mais do que a própria virulência. Estas características ou funções podem estar relacionadas ou não com mecanismos patogênicos que ocorrem no desenvolvimeto de amebíase invasiva

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasisA resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania) amazonensis de 33 kD (Larp33) foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L.) amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao antígeno total de Leishmania. A análise por imunoblot indicou que Larp33 corresponde a uma proteína de aproximadamente 40 kD expressa em promastigotas de L. (L.) amazonensis e L. (Viannia) braziliensis. Hibridização com sonda de DNA correspondente a parte do fragmento clonado, também demonstrou que o gene codificador desta proteína é expresso em promastigotas destas duas espécies. Larp33 em associação com BCG (Bacille Calmette-Guerin) foi capaz de induzir 75% de proteção em camundongos C75BL/10 e BALB/c, suceptíveis à infecção por L. (L.) amazonensis. Linfócitos dos camundongos protegidos produziram IL-2 e IFN-g em resposta a Larp33. Nossos resultados indicam que Larp33 é imunogênica para linfócitos de indivíduos vacinados com Leishvacin ® e protetora para camundongos contra a infecção por L. (L.) amazonensis

The need for daily parenteral administration is an important limitation in the clinical use of pentavalent antimonial drugs against leishmaniasis. In this study, amphiphilic antimony(V) complexes were prepared from alkylmethylglucamides (L8 and L10, with carbon chain lengths of 8 and 10, respectively), and their potential for the oral treatment of visceral leishmaniasis (VL) was evaluated. Complexes of Sb and ligand at 1:3 (SbL8 and SbL10) were obtained from the reaction of antimony(V) with L8 and L10, as evidenced by elemental and electrospray ionization-tandem mass spectrometry (ESI-MS) analyses. Fluorescence probing of hydrophobic environment and negative-staining transmission electron microscopy showed that SbL8 forms kinetically stabilized nanoassemblies in water. Pharmacokinetic studies with mice in which the compound was administered by the oral route at 200 mg of Sb/kg of body weight indicated that the SbL8 complex promoted greater and more sustained Sb levels in serum and liver than the levels obtained for the conventional antimonial drug meglumine antimoniate (Glucantime [Glu]). The efficacy of SbL8 and SbL10 administered by the oral route was evaluated in BALB/c mice infected with Leishmania infantum after a daily dose of 200 mg of Sb/kg for 20 days. Both complexes promoted significant reduction in the liver and spleen parasite burdens in relation to those in the saline-treated control group. The extent of parasite suppression (>99.96%) was similar to that achieved after Glu given intraperitoneally at 80 mg of Sb/kg/day. As expected, there was no significant reduction in the parasitic load in the group treated orally with Glu at 200 mg of Sb/(kg day). In conclusion, amphiphilic antimony(V) complexes emerge as an innovative and promising strategy for the oral treatment of VL.

Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.

Background American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody. Methodology Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples. Results The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system). Conclusions The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

Background The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil. Methodology/Principal Findings Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P, Author Summary Visceral leishmaniasis (VL) is an important public health problem in different regions of the world. It presents high lethality in human cases without suitable treatment and is considered one of the most important disorders in dogs, the main domestic reservoir of the etiological agent of VL (Leishmania infantum). Most cases of VL in Latin America occur in Brazil, and control campaigns have not shown satisfactory results. The diagnosis of human and canine infection is critical for making decisions regarding surveillance and control policies. In this work, we propose a non-invasive collection method of mucosal and epithelial cells for the molecular diagnosis of canine VL by conventional polymerase chain reaction (cPCR) and for the estimation of parasite load by quantitative real time PCR (qPCR). We used nasal, oral, and ear swabs as practical, simple, painless and fast alternatives for collecting samples. These procedures are according to the need of more simplified methods for detecting L. infantum infection by using robust diagnostic techniques such as cPCR and qPCR. Additionally, potential applications for diagnosing human VL are highlighted.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly., Author Summary Leishmania amazonensis, a member of the Leishmania mexicana complex, is the causative agent of localized cutaneous leishmaniasis (LCL) and anergic diffuse cutaneous leishmaniasis (ADCL) [1,2]. It is widely distributed throughout the Amazon basin, where it infects a wide range of terrestrial rodents and, less frequently, marsupials. Its main vector is Lutzomyia flaviscutellata (Diptera: Psychodidae) widely distributed in South America and a recent study has predicted its expansion towards South of Brazil [3]. Moreover, Lutzomyia migonei (França, 1920) can also harbor the infection of this species [4,5]. Although its transmission to man is very uncommon, L. amazonensis triggers an incurable and disseminated form of cutaneous leishmaniasis [2,6]. However, most of the mechanisms involved in L. amazonensis pathogenesis are still unknown, especially those related to surface molecules. Glycoconjugates have been extensively characterized as important for the establishment of infection as they protect the parasite from the early action of the host immune system and therefore acting as invasive/evasive strategies. Consequently, we here present the role of lipophosphoglycan (LPG) of L. amazonensis in the interaction with vertebrate and invertebrate hosts.

Background Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. Methodology/Principal Findings Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. Conclusions/Significance Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis., Author Summary Cutaneous leishmaniasis is a widespread tropical disease caused by different species of Leishmania protozoa that are transmitted by infected sandflies. Clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood. Leishmania (V.) braziliensis infection may result in a devastating disease manifestation characterized by the development of destructive lesions in the oral, nasal, and pharyngeal mucosal. Ecto-nucleotidases are enzymes that are involved in the hydrolysis of extracellular nucleotides. These enzymes have been shown to correlate with virulence of Leishmania parasites. In this work, we evaluated the ecto-nucleotidase activity of promastigotes from the twenty three different L. braziliensis isolates. We demonstrated that isolates obtained from mucosal lesions present higher levels of ecto-nucleotidase activity than those from cutaneous lesions. In addition, we show that in the murine model of cutaneous leishmaniasis, promastigote forms of parasite with higher activity induce a delayed/decreased immune response that may correlate with spreading of the parasites throughout the body. Thus, we propose that the level of ecto-nucleotidase activity of promastigotes may be a marker for the development of severe clinical forms of cutaneous leishmaniasis and also a possible target for future therapeutic intervention.

Background: Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings: KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions: LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains. publishersversion published

An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os ) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

Background We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. Methodology/Principal Findings Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P, Author Summary Visceral leishmaniasis (VL) is considered the most lethal manifestation among the diseases caused by genus Leishmania. The main control measures for VL are: diagnosis and early treatment of human cases, insecticide vector control, and euthanasia of seropositive dogs. Although these strategies have been continuously applied in Brazil, the number of VL cases has increased and this disease still poses as a serious public health problem. Belo Horizonte is an endemic urban area in Brazil and it is considered by the Ministry of Health the third most affected Brazilian metropolitan region by VL. In the context of prophylaxis, the correct diagnosis of infected dogs is critical for the VL control because dogs represent the main domestic reservoir. However, the serological diagnosis techniques used in large-scale for canine screening in Brazil have important limitations including the difficulty of diagnosing asymptomatic dogs. Molecular methods based on Polymerase Chain Reaction (PCR) and real-time PCR (qPCR) have greatly improved VL diagnosis. Based on this, we aimed to evaluate different canine clinical samples for qualitative VL diagnosis by PCR and quantification of parasite load by qPCR in dogs from Belo Horizonte. The possible implications of results are discussed with emphasis on skin and conjunctival swab samples.

Background Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis. Methods/Principal Findings We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated. Conclusions/Significance Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers., Author Summary Leishmania are unicellular microorganisms that can be transmitted to humans by the bite of sandflies. They cause a spectrum of diseases called leishmaniasis, which are classified as neglected tropical diseases by the World Health Organization. The treatment of leishmaniasis is based on the administration of antimony-containing drugs. These drugs have been used since 1947 and still constitute the mainstay for leishmaniasis treatment in several countries. One of the problems with these compounds is the emergence of resistance. Our work seeks to understand how these parasites become resistant to the drug. We studied antimony-resistant Leishmania amazonensis mutants. We analyzed gene expression at the whole genome level in antimony-resistant parasites and identified mechanisms used by Leishmania for resistance. This work could help us in developing new strategies for treatment in endemic countries where people are unresponsive to antimony-based chemotherapy. The identification of common mechanisms among different species of resistant parasites may also contribute to the development of diagnostic kits to identify and monitor the spread of resistance.

This work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii . Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10 - A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge.

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 Xakriaba 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 Xakriaba Reserve, highlighting the epidemiological diversity of ATL in this region.

Experimental vaccines to protect against visceral leishmaniasis (VL) have been developed by using BALB/c mice infected with a large (107 to 108) inoculum of parasites. Remarkably, prior literature has reported that the poor protection observed is mainly due to the high susceptibility of this strain. To determine factors inherent to mice that might abrogate vaccine-induced efficacy, the present research sought to investigate the impact of the administration of different infective inoculums of Leishmania chagasi (syn. L. infantum) in BALB/c mice, evaluating subcutaneous and intravenous routes of administration as well as parasitological and immunological parameters over different periods of time. This study shows that the injection of a highly infective inoculum in mice, through both subcutaneous and intravenous routes, results in a sustained infection. The mice developed a high parasite load in the liver; however, these values diminished over time. This result did not corroborate with the parasite load in the bone marrow and brain and proved to be expressively different in the spleen and draining lymph nodes, where the values increased over time. Mice infected with a low dose of parasites (103) showed a certain resistance against infection, based mainly on the IFN-γ and oxide nitric production. Considering all the elements, it could be concluded that the models employing high doses (107) of L. chagasi in BALB/c mice can bring about an imbalance in the animals’ immune response, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates., This study was supported by grants from Pró- Reitoria de Pesquisa from UFMG (Edital 08/2011), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; CBB-APQ-01322- 08), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; APQ-577483/2008-0), and Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT/Nano-BIOFAR), CNPq. DGV and EAFC are grant recipients of CNPq, while DMO, MAFC, and MACF are grant recipients of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly- Leishmania interaction . This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO 4 backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi ) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.

Belo Horizonte, the capital of the southeastern state of Minas Gerais, Brazil, and the fourth-largest city in the country, has the highest incidence of human visceral leishmaniasis (VL) together with a high prevalence of canine VL. The Northeast Sanitary District (NSD) of Belo Horizonte has the largest historical average of human VL cases in the metropolitan region, and is classified as a priority area for epidemiological and entomological monitoring of the disease. The objectives of the present study were to determine the seasonal variation in phlebotomine fauna and to describe the environmental situations in the NSD through characterization of peri-domiciles and application of geographical information system analysis. Entomological captures were performed every two weeks during the period July 2006 to June 2007 using HP light traps placed at 16 locations where cases of human VL had been reported in 2005. The environmental characterization of these locations was accomplished using forms and photographic images. Spatial analyses was used to determine the influence of vegetation, hydrography, altitude and pockets of poverty on the occurrence of cases of human and canine VL, and of phlebotomine vectors. A total of 633 phlebotomines belonging to the subtribes Psychodopygina and Lutzomyina were captured and, of these, 75% were identified as Nyssomyia whitmani and 11% as Lutzomyia longipalpis. The majority of the studied peri-domiciles presented inadequate hygienic conditions that would favor the development of phlebotomines. No significant correlations could be established between biogeographical aspects and either the incidence of human and canine VL or the occurrence of phlebotomines. The proximity of areas with vegetation, villages, slums and open watercourses exerted little influence on the incidence of VL. These findings reinforce the urbanization of the VL profile since the disease occurred in locations where conditions that have been classically related to its prevalence were not present. The results reported herein will be important for implementing measures against VL in the study area.

This article describes a sensitive, cheap, and easy method for assaying chemotaxic responses of Leishmania promastigotes. A gradient of the substance to be assayed was produced inside a series of commercially available capillary tubes submerged in a promastigote suspension. After an incubation period, the attractiveness of the substance under test was measured by counting the number of parasites in the capillaries in a Neubauer chamber. Different responses were detected in two strains of Leishmania amazonensis and one strain of L. chagasi after standardization of the method to assay attraction to carbohydrates. Very different responses were obtained when the test was performed using promastigotes of the same strain in two different physiological states (log and stationary phase). The stationary phase cells showed an enhanced chemotaxic capability, which can be explained by the fact that the metacyclic forms commonest in stationary phase cultures have greater mobility than other promastigotes. This method will permit studies to be made of the attractive response to different substances in Leishmania species and other trypanosomatids and facilitate characterization of the potential receptors involved in the chemotaxic response. An adaptation of the method to assay the response to repellent substances is also provided.

Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-gamma and have smaller parasite load, after 8weeks of infection. Furthermore, after 1week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.

In this study, we investigated in dogs the immunogenicity and protective immunity against Leishmania (Leishmania) chagasi infection induced by vaccination with a formulation containing the recombinant A2 protein, an amastigote specific antigen, and saponin. Vaccinated animals produced significantly increased levels of total IgG and IgG2, but not IgG1 anti-A2 antibodies, and remained negative in conventional leishmaniasis serodiagnostic methods. Significantly increased IFN-gamma and low IL-10 levels were detected in vaccinated animals before and after challenge, as compared to control animals. Importantly, while the symptoms onset appeared as early as three months after infection in most control dogs, 14 months after challenge, 5 out of 7 vaccinated dogs remained asymptomatic. Therefore, immunization with rA2 antigen was immunogenic and induced partial protection in dogs, and allowed the serological differentiation between vaccinated and infected animals, an important requirement for a canine visceral leishmaniasis (CVL) vaccine.

A population-based cross-sectional study was set up in Sabará country, Southeastern Brazil, to identify asymptomatic human visceral leishmaniasis in an urban area of low disease prevalence. Blood was collected on filter paper (n=1,604 inhabitants) and examined by indirect immunofluorescent test, enzyme-linked immunosorbent assay and immunochromatographic strip test. The prevalence rates of infection ranged from 2.4 to 5.6% depending on the test used. One year later, venous blood was collected in a subset of 226 participants (102 seropositive and 124 seronegative). The tests performed were IFAT, ELISA, rk39-ELISA, polymerase chain reaction and hybridization with Leishmania donovani complex probe. No clinical signs or symptoms of leishmaniasis were observed. Using hybridization as a reference test, the sensitivity and specificity of serology were respectively: 24.8 and 71% (ELISA); 26.3 and 76.3% (rk-39); 30.1 and 63.4% (IFAT). Due to disagreements, different criteria were tested to define the infection and hybridization should be considered in epidemiological studies. Um estudo seccional de base populacional foi desenvolvido no município de Sabará, região sudeste do Brasil, para identificar a leishmaniose visceral humana assintomática em uma área urbana de baixa prevalência da doença. Foi coletado sangue em papel filtro (n=1.604 moradores), sendo examinados pela reação de imunofluoresência indireta, ensaio imunoenzimático e teste imunocromatográfico (strip test). As taxas de prevalência da infecção variaram de 2,4 a 5,6%, dependendo do teste utilizado. Um ano depois foi coletado sangue venoso de um subgrupo de 226 participantes (102 soropositivos e 124 soronegativos). Os testes realizados foram IFAT, ELISA, rk39-ELISA, reação em cadeia da polimerase e hibridização com sonda específica para o complexo Leishmania donovani. Não foi observado nenhum sinal clínico ou sintoma de leishmaniose. Usando a hibridização como teste de referência, a sensibilidade e especificidade dos testes sorológicos foram, respectivamente: 24.8 e 71% (ELISA); 26,3 e 76,3% (rk39-ELISA); 30,1 e 63,4% (IFAT). Devido a discordâncias, diferentes critérios foram testados para definir a presença da infecção e a hibridização deveria ser considerada em estudos epidemiológicos.

In order to understand the determinants of human infection by Leishmania chagasi in an urban area, a cross-sectional population based study was conducted using molecular and serologic methods to identify infection. Participants were interviewed using a pre-coded questionnaire. Two criteria were tested to identify risk factors: Model 1- including all participants positive in hybridization by Leishmania donovani complex probe; Model 2- including all participants positive for hybridization and at least one serologic test. In Model 1, the variables associated with infection were: ownership of birds, time spent outside house between 6:00-10:00 PM and garbage not collected. In Model 2, the variables associated with infection were: family with knowledge of the vector, garbage not collected, garbage not removed or buried, ownership of birds and eroded areas in the neighborhood. The risk factors identified were associated with household conditions, presence of animals and the likelihood of contact with phlebotomine sandflies. Com o objetivo de identificar os determinantes da infecção humana por Leishmania chagasi em uma área urbana, foi realizado um estudo seccional de base populacional utilizando-se métodos moleculares e sorológicos para identificar a infecção. Os participantes foram entrevistados utilizando-se questionário pré-codificado. Dois critérios foram testados para identificar os fatores de risco: Modelo 1- incluindo todos os participantes positivos na hibridização com sonda para o complexo Leishmania donovani; Modelo 2- incluindo todos os participantes positivos na hibridização e em pelo menos um teste sorológico. No Modelo 1, as variáveis associadas à infecção foram: criar pássaros, encontrar-se fora de casa entre 18:00-22:00h e não ter o lixo coletado. No Modelo 2, as variáveis associadas à infecção foram: família conhecer o vetor, não ter o lixo coletado, lixo não removido ou queimado, criar pássaros, proximidade de áreas erodidas. Os fatores de risco identificados foram associados às condições das moradias, presença de animais e probabilidade de contato com flebotomíneos.