Human mucosal leishmaniasis: Neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1 beta, IL-23, IL-6 and TGF-beta) were detected by immunohistochemistry in ML patients. IL-17(+) cells exhibited CD4(+), CD8(+) or CD14(+) phenotypes, and numerous IL-17(+) cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.

Reappraisal of the immunopathogenesis of disseminated leishmaniasis: in situ and systemic immune response

Disseminated leishmaniasis (DL) is an emerging form of Leishmania braziliensis infection characterised by multiple cutaneous lesions on different parts of the body and a high rate of mucosal involvement. Systemic production of TNF alpha and IFN-gamma in DL patients is lower than in cutaneous leishmaniasis (CL) caused by L braziliensis, which may account for parasite dissemination due to the decreased ability to control parasite growth. In this study, the systemic and in situ immune response of DL and CL patients was characterised through evaluation of chemokine and cytokine production. In situ evaluation showed similar production of IFN gamma, TNF alpha, IL-10, transforming growth factor-beta (TGF beta), chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL11 and chemokine (C-X-C motif) ligand 10 (CXCL10) in papular and ulcerative lesions from DL as well as in ulcerated lesions from CL. Serum levels of CXCL9, a chemokine that attracts 1-cells, was higher in serum from DL than from CL These data indicate that a decrease in the type 1 immune response in peripheral blood of DL patients is due to attraction of Leishmania antigen-activated T-cells to the multiple cutaneous lesions. This may account for the absence of or few parasites in the lesions and for the development of ulcers similar to those observed in CL (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Visceral leishmaniasis and pregnancy: analysis of cases reported in a central-western region of Brazil

Objectives Because of the large number of cases of visceral leishmaniasis (VL) recorded in Brazil over the last few years, this disease has been showing characteristics different from previously known ones. We report cases of pregnant women treated for VL, describing their course and outcome and the chemotherapeutic medication used according to the clinical signs and symptoms of each patient. Study design We report five cases of pregnant women treated for VL in a central-western region of Brazil. Results No case of vertical transmission was observed, even in patients who were treated after delivery. One of the patients with a late diagnosis made after the onset of symptoms died. Thus, the treatment of VL during pregnancy reduces maternal mortality and the rate of vertical transmission of the disease, being safe and effective as long as the disease is diagnosed early. Conclusion At present, amphotericin B and its derivatives appear to be the best therapeutic option for the mother-child binomial.

Canine visceral leishmaniasis: Performance of a rapid diagnostic test (Kalazar Detect (TM)) in dogs with and without signs of the disease

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms. (C) 2008 Elsevier B.V. All rights reserved.

Chemokines and chemokine receptors coordinate the inflammatory immune response in human cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) includes different clinical manifestations displaying diverse intensities of dermal Inflammatory infiltrate Diffuse CL (DCL) cases are hyporesponsive and lesions show very few lymphocytes and a predominance of macrophages In contrast localized CL (LCL) cases are responsive to leishmanial antigen and lesions exhibit granulocytes and mononuclear cell infiltration in the early phases changing to a pattern with numerous lymphocytes and macrophages later in the lesion Therefore different chemokines may affect the predominance of cell infiltration in distinct clinical manifestations In lesions from LCL patients we examined by flow cytometry the presence of different chemokines and their receptors in T cells and we verified a higher expression of CXCR3 in the early stages of LCL (less than 30 days of infection) and a higher expression of CCR4 in the late stages of disease (more than 60 days of infection) We also observed a higher frequency of T cells producing IL-10 in the late stage of LCL Using immunohistochemistry we observed a higher expression of CCL7 CCL17 in lesions from late LCL as well as CCR4 suggesting a preferential recruitment of regulatory T cells in the late LCL Comparing lesions from LCL and DCL patients we observed a higher frequency of CCL7 in DCL lesions These results point out the Importance of the chemokines defining the different types of cells recruited to the site of the infection which could be related to the outcome of infection as well as the clinical form observed (C) 2010 American Society for Histocompatibility and Immunogenetics Published by Elsevier Inc All rights reserved

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of Sao Paulo, Brazil

Background: American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of Sao Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of Sao Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area. Results: A database containing 910 individual records of sand fly occurrence in the state of Sao Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (Nyssomyia intermedia, N. neivai, N. whitmani, Pintomyia fischeri, P. pessoai and Migonemyia migonei). The distribution of each of the six incriminated or suspected vector species of ACL in the state of Sao Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - Nyssomyia intermedia and N. neivai - are highly localized, while the other sand fly species - N. whitmani, M. migonei, P. fischeri and P. pessoai - are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL. Conclusions: The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of Sao Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of Sao Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.

Survival, Population Size, and Gonotrophic Cycle Duration of Nyssomyia neivai (Diptera: Psychodidae) at an Endemic Area of American Cutaneous Leishmaniasis in Southeastern Brazil

The survival, absolute population size, gonotrophic cycle duration, and temporal and spatial abundance of Nyssomyia neivai (Pinto) were studied in a rural area endemic for American cutaneous leishmaniasis (ACL) in Conchal, Sao Paulo State, southeastern Brazil, using mark-release-recapture techniques and by monitoring population fluctuation. The monthly abundance exhibited a unimodal pattern, with forest and domicile habitats having the highest relative abundances. A total of 1,873 males and 3,557 females were marked and released during the six experiments, of which 4.1-13.0% of males and 4.1-11.8% of females were recaptured. Daily survivorship estimated from the decline in recaptures per day was 0.681 for males and 0.667 for females. Gonotrophic cycle duration was estimated to be 4.0 d. Absolute population size was calculated using the Lincoln Index and ranged from 861 to 4,612 males and from 2,187 to 19,739 females. The low proportion of females that reach the age when they are potentially infective suggests that N. neivai has a low biological capacity to serve as a vector and that factors such as high biting rates and opportunistic feeding behavior would be needed to enable Leishmania (Viannia) braziliensis Vianna transmission. This agreed with the epidemiological pattern of ACL in southeastern Brazil that is characterized by low incidence, with isolated cases acquired principally within domiciliary habitats.

Observations on the feeding habits of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Campo Grande, an endemic area of visceral leishmaniasis in Mato Grosso do Sul, Brazil

Sand flies were captured weekly with CDC light traps from December 2003 to November 2005 in three areas of Campo Grande, in the Brazilian state of Mato Grosso do Sul. These areas incorporated two patches of remnant forest and five houses. The blood meals of engorged female sand flies were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Most (327/355) of the females analysed were Lutzomyia longipalpis, of which 66.4% reacted with human blood, 64.8% with that of birds and 8.9% with that of dogs. Females that had taken human blood predominated in the residential areas and two forest patches. The following combinations of blood were also detected for L. longipalpis in some of the samples analysed: bird + human (43.4%), bird + human + dog (6.1%). The combination bird + human + dog + pig was also found for Nyssomyia whitmani. Dogs and pigs appear to have little attractiveness for L. longipalpis. The results obtained demonstrate the eclecticism and high anthropophily of L. longipalpis and raise new questions with regard to the importance of dogs in VL epidemiology and the possible role of man as a source of infection for sand flies. (C) 2008 Elsevier B.V. All rights reserved.

Seasonal variation of Lutzomyia longipalpis (Lutz & neiva, 1912) (Diptera : Psychodidae : Phlebotominae) in endemic area of visceral leishmaniasis, Campo Grande, state of Mato Grosso do Sul, Brazil

The seasonal distribution of Lutzomyia longipalpis was studied in two forested and five domiciliary areas of the urban area of Campo Grande; MS, from December 2003 to November 2005. Weekly captures were carried out with CDC light traps positioned on ground and in the canopy inside a residual forest and on the edge (ground) of a woodland and in at least one of the following ecotopes in peridomiciles-a cultivated area, a chicken coop, a pigsty, a kennel, a goat and sheep shelter and an intradomicile. A total of 9519 sand flies were collected, 2666 during the first year and 6853 during the second. L. longipalpis was found throughout the 2-year period, presenting smaller peaks at intervals of 2-3 months and two greater peaks, the first in February and the second in April 2005, soon after periods of heavy rain. Only In one of the woodlands was a significant negative correlation (p < 0.05) between the number of insects and temperature during the first year and the climatic factors (temperature, RHA and rain) was observed. In the domiciliary areas in four domiciles some positive correlations (p < 0.05) occurred in relation to one or more climatic factors; however, the species shows a clear tendency to greater frequency (72%) in the rainy season than in the dry (28%). Thus, we recommend an intensification of the VL control measures applied in Campo Grande, MS, during the rainy season with a view to reducing the risk of the transmission of the disease. (C) 2007 Elsevier B.V. All fights reserved.

Genetic and Functional Evidence Implicating DLL1 as the Gene That Influences Susceptibility to Visceral Leishmaniasis at Chromosome 6q27

Background. Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27. Methods. Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample. Results. Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 x 10(-4) at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 x 10(-7)) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 x 10(-6) < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1. Conclusions. DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

Spatial and temporal patterns of occurrence of Lutzomyia sand fly species in an endemic area for cutaneous leishmaniasis in the Atlantic Forest region of northeast Brazil

Sand fly populations of different ecological niches in the Amaraji endemic American Cutaneous Leishmaniasis (ACL) focus of the Pernambuco Atlantic Forest region of northeastern Brazil were monitored spatiotemporally. Lutzomyia whitmani was dominant in all niches but occurred in smaller numbers in forested locations. L. whitmani was significantly less seasonal than the other species, being present throughout the year while other species were more abundant between February and April. These results suggest that L. whitmani may potentially be the principal vector of ACL in the region, even though the sand fly fauna was diverse: 88% were L.whitmani and 12% belonged to 11 other species. Two other species, L. complexa (1.3%) and L. migonei (0.8%), considered to be ACL vectors in other regions, were also present. This detailed picture of the sand fly population`s abundance and spatiotemporal distribution provides a basis for future modeling studies of forecasting sand fly activity patterns and ACL occurence.

Photodynamic therapy for American cutaneous leishmaniasis: The efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance. (C) 2011 Elsevier Inc. All rights reserved.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.