Comprehensive candidate gene analysis for symptomatic or asymptomatic outcomes of Leishmania infantum infection in Brazil.

Genetic risk factors contribute to asymptomatic versus symptomatic visceral leishmaniasis (VL) outcomes following infection with Leishmania infantum. We therefore carried out a family-based (n = 918 post-quality control fully genotyped and phenotyped individuals) candidate gene study for symptomatic VL or asymptomatic delayed-type hypersensitivity (DTH) skin test phenotypes in highly endemic neighborhoods of northeast Brazil. A total of 248 SNPs were genotyped in 42 genes selected as candidates on the basis of prior genetic, immunological, and transcriptional profiling studies. The most significant association with the VL phenotype was with SNP rs6785358 (P = 5.7e-04; pcorrected = 0.026) 3.8 kb upstream of TGFBR2, the gene encoding the type 2 receptor for transforming growth factor beta (TGFß). A second inhibitory member of the TGBß superfamily signaling pathway, SMAD7, was associated with the DTH phenotype (SNP rs7238442: P = 0.001; pcorrected = 0.051). The most significant association for the DTH phenotype was with SNP rs10800309 (P = -8.4e-06; pcorrected = 3.9e-04) situated 3.1 kb upstream of FCGR2A, the gene encoding the low-affinity IIa receptor for the Fc fragment of IgG. Overall, our results imply a role for IgG-mediated inflammation in determining DTH associated with asymptomatic infection and contribute to growing evidence that the TGFß pathway is important in the immunopathogenesis of VL.

Fine mapping under linkage peaks for symptomatic or asymptomatic outcomes of Leishmania infantum infection in Brazil.

Infection with the protozoan Leishmania infantum can lead to asymptomatic infection and protective immunity, or to the progressive and potentially fatal disease visceral leishmaniasis (VL). Published studies show host genetic background determines in part whether infected individuals will develop a symptomatic or asymptomatic outcome. The purpose of the current study was to fine map chromosome regions previously linked with risk for symptomatic (chromosome 9) or asymptomatic (chromosomes 15 and 19) manifestations of L. infantum infection. We conducted a family-based genetic study of VL and asymptomatic infection (detected by a DTH skin test) with a final post quality control sample of 961 individuals with full genotype and phenotype information from highly endemic neighborhoods of northeast Brazil. A total of 5485 SNPs under the linkage peaks on chromosomes 9, 15 and 19 were genotyped. No strong SNP associations were observed for the DTH phenotype. The most significant associations with the VL phenotype were with SNP rs1470217 (p=5.9e-05; pcorrected=0.057) on chromosome 9, and with SNP rs8107014 (p=1.4e-05; pcorrected=0.013) on chromosome 19. SNP rs1470217 is situated in a 180kb intergenic region between TMEM215 (Transmembrane protein 215) and APTX (Aprataxin). SNP rs8107014 lies in the intron between exons 26 and 27 of a 34 exon transcript (ENST00000204005) of LTBP4, (Latent transforming growth factor-beta-binding protein 4a). The latter supports growing evidence that the transforming growth factor-beta pathway is important in the immunopathogenesis of VL.

Vasorelaxant activity of 7-ß-O-glycosides biosynthesized from flavonoids.

In this work we report the vasorelaxant activity of 7-ß-O-glycosides obtained with biosynthesis of naringenin-7-ß-O-glycoside (3) and quercetin-7-ß-O-glycoside (4). These compounds were obtained from naringenin (1) and quercetin (2) glycosylation catalyzed by Beauveria bassiana ATCC 7159. Screening of the best strain as a catalyst for glycosylation was carried out and the reaction conditions established. Cultures were grown in PDSM medium for 7 days at 27 °C. After purification by reverse-phase preparative HPLC, naringenin-7-ß-O-glycoside (3) and quercetin-7-ß-O-glycoside (4) were identified by (1)H and (13)C NMR. The right position and ß-configuration of the glucose was determined through HSQC and HMBC experiments. The vasorelaxation potential of naringenin, quercetin and its glycosylated derivatives was evaluated using isolated aorta in vitro models. Interestingly, results suggest that vasorelaxation properties of naringenin, rutin and its glycosides are due to different pathways.

Feeding preferences of Lutzomyia longipalpis (Diptera: Psychodidae), the sand fly vector, for Leishmania infantum (Kinetoplastida: Trypanosomatidae).

Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in Brazil, is spread mostly by the bite of the sand fly Lutzomyia longipalpis (Lutz & Neiva). We trapped sand flies in endemic neighborhoods near Natal, Brazil, where cases of human and dog VL were documented. Amplification of species-specific cytochrome b (Cyt b) genes by polymerase chain reaction revealed that sand flies from rural and periurban areas harbored blood from different sources. The most common source ofbloodmeal was human, but blood from dog, chicken, and armadillo was also present. We tested the preference for a source of bloodmeal experimentally by feeding L. longipalpis F1 with blood from different animals. There were significant differences between the proportion of flies engorged and number of eggs laid among flies fed on different sources, varying from 8.4 to 19 (P < 0.0001). Blood from guinea pig or horse was best to support sand fly oviposition, but human blood also supported sand fly oviposition well. No sand flies fed on cats, and sand flies feeding on the opossum Monodelphis domestica Wagner produced no eggs. These data support the hypothesis that L. longipalpis is an eclectic feeder, and humans are an important source of blood for this sand fly species in periurban areas of Brazil.

Leishmania infantum chagasi in northeastern Brazil: asymptomatic infection at the urban perimeter.

Visceral leishmaniasis (VL) is endemic in large cities in Brazil, including Natal. We determined the prevalence of asymptomatic human infection with Leishmania infantum chagasi and associated environmental risks around Natal. Infection was detected by Leishmania skin test (LST) and anti-leishmanial antibodies in humans and anti-leishmanial antibodies in dogs. Amongst 345 humans, 24.6% were seropositive, and 38.6% were LST-positive. Prevalence of positive serology was similar in both sexes and across all ages. However, positive LST responses increased with age, suggesting that LST is long-lasting and cumulative. Multinomial logistic analysis showed that LST response varied with location (P = 0.007) and that males were more frequently LST-positive (P = 0.027). Indicators of lower socioeconomic status associated significantly with human infection. Furthermore, there was geographic coincidence of seropositive humans and dogs (r = 0.7926, P = 0.011). These data suggest that dog and human L. i. chagasi infection are intimately interrelated in environmental conditions associated with low income.

Leptospirosis in a subsistence farming community in Brazil.

Leptospirosis has been reported in rural areas of Brazil. However, there is limited information about the exposure risk or the risk of Leptospira infection for rural-based populations. A cross-sectional study was carried out in order to determine the prevalence and risk factors for prior Leptospira infection in a rural subsistence farming region of the state of Rio Grande do Norte, an area in which outbreaks of leptospirosis have occurred. Among 290 individuals enrolled, 44 (15.2%) had anti-Leptospira IgM antibodies as determined by IgM ELISA. Infection tended to occur with activities related to the rice fields (P=0.08). Our findings indicate that Leptospira infection occurs even in years of low rainfall, and may have an important impact among poor rural-based subsistence farmers in Brazil. Additional studies are needed to characterize the mode of transmission in this region.

Leishmania chagasi T-cell antigens identified through a double library screen.

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.

An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil.

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n = 135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n = 390); Ab +: individuals with negative DTH response and seropositive (n = 21); DTH -: individuals with negative DTH and seronegative (n = 560). The mean +/-SD age of VL was 9.3+/-12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.

Changing epidemiology of American cutaneous leishmaniasis (ACL) in Brazil: a disease of the urban-rural interface.

American cutaneous leishmaniasis (ACL) was first reported from the south-western region of the state of Rio Grande do Norte in 1987. São Miguel municipality and adjacent areas have accounted for 90% of the American cutaneous leishmaniasis cases in the region since then. A population survey conducted in São Miguel and adjacent areas was undertaken to identify individuals with a history of ACL. The incidence of ACL, distribution by age and gender, clinical characteristics, family clustering, relationship to animals, association with skin test responses, and development of mucosal lesions were determined. Males and females were equally likely to be infected by Leishmania. Thirty-eight percent of the individuals tested were found to be Montenegro skin test positive. No difference in the Montenegro skin test was observed with gender, but the response increased with age. Among the 140 ACL cases treated in a 4-year period in São Miguel, five (3.6%) developed mucosal involvement. Sandflies capable of transmitting Leishmania were captured inside households and in the areas surrounding them. The high density of sandfly species potentially able to transmit Leishmania species and the age distribution of cases of cutaneous leishmaniasis suggest that transmission of the parasite occurred inside or in close proximity to houses.

The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection.

The sensitivity and specificity of a Leishmania chagasi recombinant K39 (rK39)-based enzyme-linked immunosorbent assay (ELISA) for visceral leishmaniasis (VL) was assessed in Natal, Brazil. Anti-rK39 antibodies were detected in 93.3% of patients with parasitologically confirmed VL (n = 120) and in 33 others with clinically diagnosed disease. Anti-rK39 antibodies decreased significantly following treatment. The presence of antibodies was inversely correlated with development of a positive leishmanin skin test result. Anti-rK39 antibodies were detected in only 2.9% of asymptomatic subjects with a positive skin test result (n = 168). They were not detected in healthy controls (n = 30) or in persons with Chagas' disease (n = 13) or active tuberculosis (n = 31). Antibodies were found in only one of 13 patients with cutaneous leishmaniasis. In contrast, an ELISA using total L. chagasi promastigote antigen was sensitive, but not specific. The results indicate that the rK39-based ELISA is a sensitive and specific diagnostic test for symptomatic VL and can differentiate progressive from self-resolving infection.