Porto de Galinhas, Pernambuco, Brazil, May 13 th to 17th , 2013 Enotel
Abstract Book
Porto de Galinhas, Pernambuco, Brazil, May 13th to 17th, 2013 Enotel www.worldleish5.org
Abstract Book
Brazil – Pernambuco – Ipojuca
Fifth World Congress on Leishmaniasis
WorldLeish5 13th to 17th May 2013 Porto de Galinhas, Pernambuco, Brazil The World Congress on Leishmaniasis is the most important meeting on a disease of high priority in the World Health Organization’s list of Tropical Neglected Diseases. The leishmaniases are going to be around for a very, very long time because almost all of them have an animal reservoir, which excludes the possibility of elimination. It is therefore essential to define guidelines targeting the control. WL5 gathers leishmaniacs from all around the world. Unlike other meetings it does not focus on one academic or public health field but on one group of diseases – the leishmaniasis. Research on Leishmania parasites and the diseases they cause has increased over the last decade. Indeed, they have proven to be exciting models for studying parasite-host and vector-host interactions, immunological mechanisms, molecular biology, genetics, epidemiology and other related areas. The principal aims of WorldLeish are to foster interaction between leishmaniacs from around the world and to provide a unique forum for discussing the state-of-the-art of leishmaniasis research, including the perspectives related to the control and prevention. Besides this it allows participants to share their most recent findings and to meet and interact with some of the world’s leading experts. The first World Congress on Leishmaniasis (WL1) was held in Istanbul, Turkey, in 1997 and was a great success. WL2 was held in Crete, Greece, in 2001, WL3 took place in Sicily, Italy in 2005 and WL4 took place in 2009 in Lucknow, India. The meeting’s success and popularity continued to increase as did the number of participants. However, they all took place in the Old World which made it difficult for younger American leishmaniacs to attend. Now, after three meetings in Europe and one in Asia WorldLeish is finally taking place in an American country. Brazil represents one of most important leishmaniasis endemic areas, with the occurrence of many varieties of parasites which cause different clinical forms, including cutaneous, mucocutaneous and visceral leishmaniasis. Brazil has one of the world’s biggest critical masses of scientists and public health authorities working on leishmaniasis. This is due to the ever increasing importance of the disease and the levels of funding from different federal and state governmental bodies.
WL5 has broken all previous records. A total of 1,426 abstracts have been received which exceeded by far the number that was expected. Although a pleasant surprise, this created certain logistical problems. The challenge was how to accommodate so many high quality contributions as oral presentations since the number of sessions was limited. So, some had to be changed to posters. To help in analyzing these abstracts the Organizing Committee counted on both the International Scientific Committee and the Brazilian Scientific Committee, composed of 52 Brazilian leishmaniacs. The final timetable has 5 simultaneous daily sessions with 1 satellite symposium, which to date is the most extensive WL program ever. Our actual Program introduces, besides 43 oral sessions and 14 Satellite Symposia, 22 special sessions designated as Hot Spots. The subjects for the Hot Spots are those that need greater focus or are considered to be controversial according to a survey performed on Leish-L. Abstracts in these specific areas were selected for presentation in the relevant Hot Spot. A total of 962 posters are presented in 3 evening sessions and specially selected posters will be located in 6 different areas, that are being called “Thinking Boxes”, to serve as discussion canters. The number of participants has broken all records. Approximately 1,200 individuals from all walks of science and public health will be attending. We are grateful to the enthusiasm of all who have guaranteed WL5´s success, especially the younger ones from Latin America for whom this is possibly a once in a lifetime opportunity to attend a WL meeting. We want to acknowledge our deepest gratitude to the organizations that have generously supported WL5 financially: Health Surveillance Secretary/Ministry of Health/Brazil, Oswaldo Cruz Foundation, Bill & Melinda Gates Foundation, Belgian Development Cooperation, NIH, PAHO/WHO, CNPq, CAPES, DECIT, FACEPE, and the Sponsorship of BAYER, SANOFI, ZOETIS/PFIZER, HERTAPE CALIER, PALADIN, BIOMANGUINHOS and MSD. Without their funding WL5 would probably not have taken place. Due to committee’s dedication together with the generosity of funding agencies WL5 has been able to award registration waivers that include help with accommodation to 37 applicants selected from 15 countries were funds are scarce and where the disease is a significant public health issue. We have put together a scientific program that has something for everybody and hopefully will open new horizons for both young and old leishmaniacs. We also wish that all of you enjoy the tranquility of Porto de Galinhas’s warm and clear waters, together with its excellent variety of local and international cuisine. These settings will create the perfect atmosphere for making new friends and reuniting with old ones. Have a wonderful time!
The Organizing Committee
ABSTRACTS -ORAL-
- CELLULAR AND MOLECULAR BIOLOGY [117] O 001 - MUTATIONAL ANALYSIS OF AN ESSENTIAL PROTEIN: LEISHMANIA DONOVANI HSP90 HOMBACH, A.L.1; CLOS, J.1; WIESE, M.2 1.BERNHARD-NOCHT INSTITUT FOR TROPICAL MEDICINE, HAMBURG, GERMANY; 2.2STRATHCLYDE INSTITUTE OF PHARMACY AND BIOMEDICAL SCIENCES, GLASGOW, UNITED KINGDOM.
Keyword:leishmania donovani; hsp90; phosphorylation
Abstract: Antje Hombach1, Martin Wiese2 and Joachim Clos1 1Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany and 2Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS),University of Strathclyde, Glasgow, UK There is compelling evidence that the L. donovani heat shock protein 90 (LdHsp90) plays a crucial role in life cycle control and that it is essential for the parasite's viability [1]. Hsp90 is also the target for stage-specific phosphorylation [2]. So far, the essential nature of Hsp90 and the large gene copy number in Leishmania have precluded mutational analysis of this important protein. However, pharmacological inhibition of Hsp90 has been feasible using specific inhibitors such as geldanamycin and radicicol (RAD). Interestingly, the Hsp90 from the RAD-producing fungus Humicola fuscoatra has a low binding affinity for RAD, caused by a single leucine to isoleucine exchange in the highly conserved ATP binding pocket [3]. Indeed, we can show that a LdHsp90-L33I (Hsp90rr) transgene also mediates pronounced resistance against RAD in vitro whilst overexpression of wild type Hsp90 has no such effect [4]. This creates a system in which growth and/or infectivity of parasites under RAD inhibition depends on the functionality of Hsp90rr transgenes, whilst endogenously coded protein is inactivated. Employing this strategy, we find that a C-terminal putative binding motif (MEQVD) for the co-chaperone Sti-1 is critical for Hsp90 function, and for proliferation of the both life cycle stages. [4] We also used this system to investigate the role of LdHsp90 phosphorylation in both life cycle stages of L. donovani. By introducing mutations into putative phosphorylation sites in LdHsp90rr we aim to assess the impact of Hsp90 phosphorylation on either promastigote or amastigote stage and thus establish a link between the signal transduction pathways and the Leishmania chaperone system. To date, seven putative phosphorylation sites have been analysed. Three phosphorylation sites in LdHsp90 appear as important for promastigote growth in vitro whilst three other sites contribute to intracellular proliferation of amastigotes in bonemarrow-derived macrophages. We aim to analyse the impacts of other putative phosphorylation sites in both life cycle stages and to identify the responsible protein kinases. 1. Wiesgigl, M. and J. Clos, Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani. Mol Biol Cell, 2001. 12(11): p. 3307-16. 2. Morales, M., et al., Phosphoproteome dynamics reveals heat shock protein complexes specific to the Leishmania infectious stage Proc Natl Acad Sci U S A, 2010. 107(18): p. 8381-8386. 3. Prodromou, C., et al., Structural basis of the radicicol resistance displayed by a fungal hsp90. ACS Chem Biol, 2009. 4(4): p. 289-97. 4. Hombach, A., et al., The Hsp90-Sti1 interaction is critical for Leishmania donovani proliferation in both life cycle stages. Cellular microbiology, 2012.
- CELLULAR AND MOLECULAR BIOLOGY [206] O 002 - FUNCTIONAL ANALYSIS OF EPISOMES IN LEISHMANIA DONOVANI MANNAERT, A.1; DUMETZ, F.1; IMAMURA, H.1; VANAERSCHOT, M.1; BERRIMAN, M.2; SUNDAR, S.3; KHANAL, B.4; RIJAL, S.4; DUJARDIN, J.1
1.INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM; 2.WELLCOME TRUST SANGER INSTITUTE, HINXTON, UNITED KINGDOM; 3.BARANAS HINDU UNIVERSITY, VARANASI, INDIA; 4.B.P. KOIRALA INSTITUTE OF HEALTH SCIENCES, DHARAN, NEPAL.
Keyword:episome; transfection; map kinase 1
Abstract: Amplification of whole chromosomes or genes to increase gene dosage in the absence of transcriptional control is well established in Leishmania. An increase in gene copy number can be accomplished by tandem duplications, or by the formation of extra-chromosomal episomes through homologous recombination between repeats. Two circular episomes have been identified in a population of 17 drug resistant and sensitive clinical strains of Leishmania donovani from the Indian subcontinent by whole genome sequence analysis. One episome is the H-locus, which contains the resistance-associated genes MRPA, an ABC thiol transporter, and the terbinafine resistance locus, and is already known to appear in experimental conditions such as induction of drug resistance. The other episome is recently described and carries a mitogenactivated protein kinase homolog (MAPK1) and a secreted acid phosphatase gene (SAcP), both regulatory proteins that are part of signaling pathways triggered by environmental changes. In an extended dataset with >100 clinical L. donovani lines, both episomes were found in 94% of the lines and their copy number is linked with antimonial (SSG) treatment failure in the corresponding visceral leishmaniasis patients. The episomes are stable during promastigote growth stages, but amastigotes appear to have higher copy numbers. MAPK1 plays an important role in processes such as proliferation, differentiation, stress response and apoptosis, and appears to be essential for amastigote survival and proliferation in the mammalian host. Moreover, higher expression levels of this gene in SSG-resistant laboratory strains of L. donovani suggest that it is also involved in drug resistance. To determine the significance of the episomal amplification of MAPK1 and the other genes, a functional analysis is done by overexpression of the episomes and the separate genes. A Nepalese strain in which both episomes are naturally absent is transfected with the MAPK1- and MRPA-episome and with the episome genes. The effect of the transfections on the parasites’ fitness is assessed by determination of phenotypic traits such as metacyclogenesis rate, infectivity and stress tolerance. In addition, a kinase assay is performed to determine whether any of the episomally amplified genes is a target of MAPK1. - CELLULAR AND MOLECULAR BIOLOGY [234] O 003 - THE FAMILY OF LEISHMANIA MEXICANA A600 PROTEINS PLAY AN ESSENTIAL ROLE IN ANAEROBIC ENERGY METABOLISM OF AMASTIGOTE CELLS. MARR, A.K.; ABRAHAM, T.; COHEN, S.; LOUTET, M.; MCMASTER, R.W. UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, CANADA.
Keyword:mitochondria; anaerobic respiration; fumarate reductase
Abstract: Differentiation of Leishmania species from the promastigote- to the amastigote stage involves significant morphological and biochemical changes in the parasite. However, most genes of the leishmania genome are constitutively expressed in both life stages. The underlying molecular mechanisms that are responsible for these morphological and biochemical changes are mediated by differential gene expression and thus are likely mediated at the functional protein level. An exception is the Leishmania specific A600-gene family (genes designated as A600.1, A600.2,
A600.3 and A600.4) of as yet unknown function. These genes are up-regulated in the amastigote stage, and are essential for parasite replication in host macrophage cells. In this study, we set out to characterize further the function of the A600 proteins. Given their stage-specific expression and potential role in virulence, an A600-deficient mutant may have a potential role as a vaccine candidate for leishmaniasis. To determine the subcellular localization of the A600 proteins, we generated two fusion constructs, A600.1-GFP and A600.4-RFP, and following transfection these fusion proteins were expressed in L. mexicana wild type cells. Using epifluorescence-, confocal- and electron microscopy, we demonstrate that both proteins are localized in the inner mitochondrial membrane of L. mexicana promastigotes. To test whether these proteins play a role in cell respiration, we quantitated the ATP concentration and the redox potential in promastigote and amastigote cells of a wild type L. mexicana strain and an A600 deletion mutant, respectively. We show that promastigotes of wild type and mutant cells have similar ATP concentration and similar redox potential. However, ATP concentration in A600 deletion amastigotes is significantly lower and redox potential impaired compared to wild type amastigotes. Furthermore, the redox poteintial in wild type amastigote but not A600 deletion amastigotes can be significantly reduced using LicochalconeA, a fumarate reductase specific inhibitor. In summary, our results suggest a role of A600 proteins in the anaerobic respiratory chain of amastigotes potentially by contributing to the fumarate reductase activity.
- CELLULAR AND MOLECULAR BIOLOGY [235] O 004 - REGULATION OF LEISHMANIA AQUAGLYCEROPORIN AQP1 MUKHOPADHYAY, R.1; MANDAL, G.1; SHARMA, M.1; TAMAS, M.J.2; WIESE, M.3; BHATTACHARJEE, H.1 1.FLORIDA INTERNATIONAL UNIVERSITY, MIAMI, UNITED STATES; 2.UNIVERSITY OF GOTHENBURG, GOTHENBURG, SWEDEN; 3.UNIVERSITY OF STRATHCLYDE, GLASGOW, UNITED KINGDOM.
Keyword:mapk; aquaglyceroporin; utr
Abstract: The Leishmania aquaglyceroporin, AQP1 is responsible for important physiological functions such as volume regulation and osmotaxis as well as drug (trivalent antimony, SbIII) sensitivity. However, the mechanism of regulation is largely unknown. Since LmMAPK2 complemented the function of yeast p38 MAPK (Hog1p) which negatively regulated yeast aquaglyceroporin and MAPK2 mRNA was upregulated under all stresses in Leishmania, a close interplay of AQP1 and MAPK2 was hypothesized. Co-overexpression of MAPK2 and AQP1 led to SbIII hypersensitivity as compared to only AQP1 or MAPK2 overexpressing parasites. Only LmMAPK2 overexpressing cells were also hypersensitive to SbIII compared to vector control whereas ΔMAPK2 promastigotes became resistant. Thr197, a putative phosphorylation site of LmAQP1 when changed to alanine abrogated this phenotype suggesting that a phosphorylation event was responsible. Expression studies revealed that phosphorylation of LmAQP1 by MAPK2 at T197 stabilized the protein. The AQP1 U-rich 3’UTR contains several ARE (AU rich sequences) and CURE (CU rich sequences) motifs that regulate mRNA stability in higher eukaryotes. The stability studies revealed that AQP1 mRNA is somewhat unstable. Taken together, these data suggest that MAPK2 functions as a positive regulator of LmAQP1 at the post-translational level. The mechanisms of this regulation and the role of different controlling elements in the 3’ UTR along with the trans-acting factors will be discussed. - CELLULAR AND MOLECULAR BIOLOGY
[383] O 005 - LEISHMANIA CO-OPT THE HOST CELL’S COPII PROTEIN EXPORT MACHINERY TO EXPORT LEISHMANIA DERIVED PROTEINS FROM THE LEISHMANIA PARASITOPHOROUS VACUOLE INTO THE HOST CELL CYTOSOL. KIMA, P.E. UNIVERSITY OF FLORIDA, GAINESVILLE, UNITED STATES.
Keyword:parasitophorous vacuole; copii vesicles; export
Abstract: Once inside mammalian cells, Leishmania can modulate host cell responses to various stimuli. However, it is not known how Leishmania proteins are exported from the membrane bound compartment called Leishmania parasitophorous vacuoles (LPVs) in which they reside within infected cells. Studies of other intra-vacuolar pathogens have shown that molecules synthesized within those pathogens are exported through intricate export machineries across the vacuolar membrane to the cell cytosol where they exercise their effects on the infected cell. There is now evidence that LPVs interact continuously with the early secretory pathway (ESP) of the host cell, which result in the display of ESP molecules in LPVs. These ESP molecules include Sar1a, sec23, and sec24, which are molecular components of COPII vesicles. We investigated the likelihood that host cell COPII molecules on LPVs can form COPII vesicles that bud off LPVs loaded with Leishmania derived molecules. Presently, only a handful of Leishmania molecules have been shown to be exported out of LPVs into the host cell cytosol; however, the mechanism(s) by which those and other Leishmania parasite molecules are exported from LPVs is not known. We provide evidence here that the intracellular variant of Tryparedoxin peroxidase is indeed exported from LPVs in COPII vesicles. Expression of a dominant negative sar1a molecule that blocks the formation of COPII vesicles also blocks the export of Tryparedoxin peroxidase into the infected cell cytosol. These studies show for the first time that Leishmania co-opt a host cell protein export machinery for the export of their proteins from the LPV into the host cell cytosol where they can target host cell processes. - CELLULAR AND MOLECULAR BIOLOGY [594] O 006 - A SMALL HEAT SHOCK PROTEIN IS ESSENTIAL FOR LEISHMANIA SURVIVAL AT THE TEMPERATURES OF THE MAMMALIAN HOST HOMBACH, A.; OMMEN, G.; CLOS, J. BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE, HAMBURG, GERMANY.
Keyword:small hsp; temperature tolerance; geldanamycin
Abstract: Since the 1980s there was speculation that expression of heat shock proteins (Hsps) at elevated temperatures was responsible for an induced temperature tolerance required for the survival of Leishmania amastigotes in the mammalian host [1]. However, the constitutive abundance of the major heat shock proteins, Hsp70 and Hsp90 [2], and the moderate impact of Hsp100 on thermotolerance [3] did not support this concept. In plants and extremophiles, thermotolerance is largely mediated by small Hsps of the Hsp20 family [4]. Related to these are the co-chaperones of the P23 family. Both families, in turn, share a highly conserved signature domain with the eye lens α-crystallins of higher eukaryotes. We have identified two members of the α-crystallin superfamily of proteins in the L. infantum genome database and we used gene replacement analysis to determine their role and impact in L. donovani.
As promastigotes, P23 replacement mutants are viable with minor growth deficiencies. Similar to its yeast ortholog, P23 replacement causes increased sensitivity towards the Hsp90 inhibitor geldanamycin. Infectivity in bone marrow-derived macrophages is reduced by half for the P23-/mutants, a defect that can be eliminated by episomal P23 gene copies. P23-/- mutants show a wild type-like growth at elevated temperatures (37°). Hsp20, by contrast, shows a noticeable growth inhibition at 25°C and is indispensable for L. donovani promastigote survival at temperatures exceeding 34°C. Consequently, the loss of Hsp20 also abolishes L. donovani survival within ex-vivo macrophages. Again, expression of episomal transgene restores high temperature viability and infectivity to the Hsp20-/- mutants. Lack of Hsp20 does not affect geldanmycin sensitivity, arguing against a role as co-chaperone in Hsp90 foldosome complexes. Currently, we aim at establishing the expression kinetics for both P23 and Hsp20, as well as their subcellular localisation in both life cycle stages. We also plan to follow up on preliminary data that indicate a presence of Hsp20 and P23 in L. donovani exosomes and report on the results. 1. Hunter, K.W., C.L. Cook, and E.G. Hayunga, Leishmanial differentiation in vitro: induction of heat shock proteins. Biochem Biophys Res Commun, 1984. 125(2): p. 755-60. 2. Brandau, S., A. Dresel, and J. Clos, High constitutive levels of heat-shock proteins in humanpathogenic parasites of the genus Leishmania. Biochem J, 1995. 310(Pt 1): p. 225-32. 3. Hubel, A., et al., Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite. Mol Cell Biol, 1997. 17(10): p. 5987-95. 4. Haslbeck, M., et al., Some like it hot: the structure and function of small heat-shock proteins. Nature structural & molecular biology, 2005. 12(10): p. 842-6.
- GENETICS, EVOLUTION AND TAXONOMY [870] O 007 - THE TAXONOMIC STATUS OF THE SHANNONI COMPLEX IN BRAZIL (DIPTERA, PSYCHODIDAE, PHLEBOTOMINAE) SÁBIO, P.B.; DE ANDRADE, A.J.; GALATI, E.A.B.
DEPARTAMENTO DE EPIDEMIOLOGIA, FACULDADE DE SAÚDE PÚBLICA, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:phlebotominae; shannoni complex; psathyromyia
Abstract: The Shannoni complex of the genus Psathyromyia (Diptera, Psychodidae) is constituted by several species with great morphological and morphometric similarities which have led to erroneous identifications of the taxa. This complex has been implicated as vector of Leishmania spp. and trypanosomatids. The study sought to assess the status of the species of this complex, by means of their morphological and morphometric characters; associated with the complex. The specimens mounted on slides were analyzed morphologically, the initial identification given on the label of each slide being ignored. Later they were separated into groups of the same species and then measurements were taken to test the similarity or difference of or between the groups, using ANOVA. The results showed that Pa. bigeniculata and Pa. limai are valid species, however, the male of Pa. limai had erroneously been associated with the female
holotype, the male being in fact that of Pa. bigeniculata, which presents wide distribution in Brazil and has hitherto been identified as Pa. shannoni. The male corresponding to Pa. limai is in fact described as that of Pa. pestanai, the female of these species being morphologically identical. A taxon which occurs in the upper Ribeira Valley is a species unknown to the science. The results confirm the validity of Pa. pifanoi, but show that Pa. cuzquena is a junior synonym of this. The taxon which has been being erroneously identified, in some of the states of the Brazilian federation, as Pa. abonnenci, is morphologically closer to Pa. microcephalla, which seems to be a valid species, though it is actually classified as a junior synonym of Pa. shannoni. As a matter of fact, Pa. abonnenci is a valid species distinct from the taxon similar to Pa. microcephalla. The complex Shannoni consists of seven valid species and three synonyms: Pa. shannoni, Pa. limai (= Pa. pestanai), Pa. bigeniculata (= Pa. limai, macho), Pa. microcephalla, Pa. pifanoi (= Pa. cuzquena), Pa. abonnenci and Psathyromyia sp. nov. Further study on Pa. microcephalla and its true distribution is called for. The correct identification of these taxa will enable understanding the actual epidemiological role of the species of this complex as vectors of Leishmania spp. Financial support: CAPES; FAPESP. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [1341] O 008 - ENVIRONMENTAL FACTORS ASSOCIATED WITH CUTANEOUS LEISHMANIASIS IN AN ANDEAN FOCUS IN COLOMBIA OCAMPO, C.B.1; VALDERRAMA, C.H.2; FERRO, M.C.3; PEREZ, M.1; MUNSTERMANN, L.4; QUINNELL, R.5; ALEXANDER, N.1
1.CIDEIM, CALI, COLOMBIA; 2.UNIVERSIDAD ICESI, CALI, COLOMBIA; 3.INSTITUTO NACIONAL DE SALUD, BOGOTA, COLOMBIA; 4.YALE UNIVERSITY, NEW HAVEN, UNITED STATES; 5.LEEDS UNIVERSITY, LONDON, UNITED KINGDOM.
Keyword:leishmaniasis ; lutzomyia; ecology
Abstract: Environmental and ecological risk factors for cutaneous leishmaniasis (CL) were investigated at local and landscape scales during one of the largest outbreaks recorded in Colombia, with a peak incidence of 6202 per 100,000 inhabitants in 2004. The high proportions of infected children (36.5% of 2,835 cases) and women (35.5% of 1,811 adults) suggested domestic transmission. Landscape scale analysis was carried out using remote-sensed data in all the affected townships. In a spatial model, predictor variables included land use, elevation, and climatic variables such as mean temperature and precipitation. Local analysis examined peridomestic habitats, comparing a township with high prevalence of CL (74% of 172 inhabitants) and a township with low prevalence (1.3% of 354). Habitat composition was evaluated in a 100m radius around the house and sand fly abundance quantified using CDC traps. Landscape analysis showed that incidence of cutaneous leishmaniasis was independently associated with higher coverage with forest or shrubs (2.6% greater for each additional percent coverage, 95% CI 0.5-4.9%), and lower population density (22% lower for each additional 100 people per square kilometer, 95% CI 7-41%). This corresponds with the findings at “local scale” were the high transmission township had higher coverage of forest (23 versus 8.4%) and shade coffee (30.7 versus 11%), and less coffee monoculture (16.8 versus 26.2%) and pasture (6.3 versus 12.3%), compared to the low transmission township. Studies at both the local and landscape scales demonstrated that altitude (>1300m) and land use (forest coverage) are risk factors for infection during this epidemic. At the local scale there was a significant negative correlation between the abundance of the vector Lutzomyia longiflocosa inside and around the house and the distance of the house to dense vegetation, suggesting that such habitats are important for daytime resting and/or breeding sites. The results from these studies endorse the use of remote-sensed variables such as altitude and land use as proxy measures of vector abundance, to identify areas at high risk of Leishmania transmission.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [1034] O 009 - PHLEBOTOMINE SAND FLY FAUNA IN AN AREA OF VISCERAL LEISHMANIASIS ENDEMICITY WHERE LUTZOMYIA LONGIPALPIS IS ABSENT DANTAS PEREIRA, P.D.1; DA SILVA, F.J.1; BRANDÃO-FILHO, S.P.1; DANTAS-TORRES, F.2
1.DEPARTMENT OF IMMUNOLOGY, AGGEU MAGALHÃES RESEARCH CENTER, OSWALDO CRUZ FOUNDATION, RECIFE, PE, BRAZIL; 2.FACOLTÀ DI MEDICINA VETERINARIA, UNIVERSITÀ DEGLI STUDI DI BARI, VALENZANO, ITALY.
Keyword:fauna; phlebotomine; visceral leishmaniasis
Abstract: Visceral leishmaniasis is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sand flies of the genus Lutzomyia in the New World. The Brazilian phlebotomine sand fly fauna is composed of over 230 species. The primary vector of L. infantum in Brazil is Lutzomyia longipalpis. However, cases of visceral leishmaniasis have sporadically been reported in areas where the primary vector has never been found. The aim of this study was to provide assess the phlebotomine sand fly fauna in Vicência (Atlantic rainforest region of Pernambuco, north-eastern Brazil), an endemic area of visceral leishmaniasis endemicity where L. longipalpis is thought to be absent. From August to December 2004 and from February to November 2005, phlebotomine sand flies were monthly collected, during three consecutive nights, using CDC light traps and a Shannon trap. Each night, a total of 10 traps were placed inside houses, near animal shelters and remnants of Atlantic rainforest. They were placed about 1 m above the ground and operated from 5:00 pm to 6:00 am. Phlebotomine sand flies were kept in vials containing 70% ethanol until mounting and identification. A total of 37,419 phlebotomine sand flies belonging to 21 species and two genera were collected. Lutzomyia whitmani (n=29,817) was the most abundant species, followed by Lutzomyia migonei (n=3,493), which together represented 89% of the collections. Males (n=27,349) predominated over females (n=10,070), with a overall sex ratio (male:female) of 2.7:1. Three remarkable peaks were observed during the study period. In October 2004, there was a peak determined by an increase in number of L. whitmani and L. complexa specimens collected. There was an increase in the number of L. complexa, L. evandroi, L. migonei and L. whitmani specimens collected in April 2005. Furthermore, the L. evandroi population increased in May 2005. In conclusion, this study confirmed the absence of L. longipalpis and adds weight to the hypothesis on the participation of L. migonei in the transmission of L. infantum in this area of north-eastern Brazil. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [974] O 010 - SPECIES COMPOSITION AND RELATIVE ABUNDANCE OF PHLEBOTOMINE SAND FLIES IN DIFFERENT HABITATS IN SOUTHERN ITALY DANTAS-TORRES, F.1; TARALLO, V.D.2; LIA, R.P.2; OTRANTO, D.2
1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSITÀ DEGLI STUDI DI BARI, VALENZANO, ITALY.
Keyword:sand flies; ecology; italy
Abstract: The epidemiology of visceral leishmaniasis is strongly influenced by host-associated factors, and by the presence and abundance of competent phlebotomine sand fly vectors. Nonetheless, data on factors driving phlebotomine sand fly ecology are still meagre, even in endemic areas. This lack of knowledge might hamper the implementation of control measures in risk areas. The present study was conducted in Basilicata, southern Italy, where visceral leishmaniasis is endemic. Our objectives were to assess the species composition and relative abundance of phlebotomine sand flies in six different habitats: a stone wall near a woodhouse (CS1); a tree
near a volcanic rock (CS2); a tree in a meadow habitat (CS3); a sheep stable (CS4); a chicken coop (CS5); and the garage of a house (CS6). In total, six species were identified (i.e., Phlebotomus perfiliewi, Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus papatasi, Phlebotomus mascitti, and Sergentomyia minuta), corresponding to 75% of the total number of phlebotomine sand fly species found in Italy. Phlebotomus perfiliewi was the dominant species, representing 87.49% of the collections, followed by P. perniciosus (9.92%). Phlebotomus mascitti was an uncommon species, with only one specimen collected. The greatest species richness and abundances were found in CS6 (4 species, 9,066 specimens), CS5 (5 species, 2,228 specimens) and CS4 (5 species, 1,375 specimens). The lowest number of phlebotomine sand fly specimens collected was recorded in CS2, which was located in a high altitude area. On the other hand, highest abundances and greatest species richness were recorded in sites near or in human houses and they were always associated with the presence of domestic animals, such as sheep, goats, chickens, dogs and cats. Our findings confirm a high level of species richness in southern Italy and indicate that the phlebotomine sand fly vectors are highly adapted to human dwellings. Furthermore, data here presented indicate that the presence of domestic animals might be a risk factor for the establishment of vector species (e.g., P. perfiliewi) nearby or in human houses. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [923] O 011 - EXPANSION OF THE ALTITUDINAL DISTRIBUTION OF LUTZOMYIA LONGIPALPIS (LUTZ & NEIVA, 1912) (DIPTERA: PSYCHODIDAE) IN THE CENTRAL ANDEAN REGION OF COLOMBIA: POTENTIAL RISK OF NEW FOCI FOR VISCERAL LEISHMANIASIS. ACOSTA, L.A.1; MONDRAGON, K.1; VERGARA, D.2; VELEZ, A.1; CADENA, H.1; CARRILLO, L.M.3; VELEZ, I.D.1 1.PECET, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA; 2.INDIANA UNIVERSITY, BLOOMINGTON, UNITED STATES; 3.PECET, AGRARIAN SCIENCES, UNIVERSITY OF ANTIOQUIA, MEDELLIN, COLOMBIA.
Keyword:sandfly vector; lu. longipalpis; risk factors
Abstract: The main vector of visceral leishmaniasis in Latin America is Lutzomyia longipalpis, which has been considered to be a complex of species with wide distribution throughout the continent. It exhibits adaptation to different habitats, and several studies suggest that climate variations may affect the vector’s biology, and the increase in the spatial distribution, including the urbanization. This leads to the appearance of new foci and the emergence of the disease. The objective of this research was to conduct entomological surveillance in the area of influence of the Hydroelectric Plant Miel I, in the Caldas province in Colombia. In 1996 entomological surveillance in the municipalities of La Dorada, Marquetalia, Samana and Norcasia, located at the Magdalena river valley, was performed in a transect of approximately 400 km at an altitude range of approx. 200 to 1800 masl, sticky traps were used for the capture of sandflies. In this transect, Lu longipalpis samples were found in La Dorada at 350 masl. The sandflies were identified using the taxonomical key of Young and Duncan (1994). Ten years later, the study was repeated in the same transect, in this opportunity Lu longipalpis was found again at an altitude of 392 masl, and for the first time in Marquetalia at 1387 masl, which is the maximum altitude ever reported for this species in the country. We suggest that the increase in the geographical and altitudinal distribution could be related to environmental factors such as the rise in temperature due to global climate change which promotes the establishment of Lu. Longipalpis in new environments, generating new epidemiological risk for new visceral leishmaniasis foci in the country.
- CONTROL PROGRAMS [1074] O 012 - STUDY OF THE INFLUENCE OF ENVIRONMENTAL AND LIVING CONDITIONS ON THE POPULATION DENSITY OF SAND FLY P.ARGENTIPES AT THE HOUSEHOLD LEVEL IN INDIAN VILLAGES MALAVIYA, P.1; HASKER, E.2; SINGH, R.P.1; GEERTRUYDEN, J.V.3; BOELAERT, M.2; SUNDAR, S.1 1.BANARAS HINDU UNIVERSITY, VARANASI, INDIA; 2.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 3.ANTWERP UNIVERSITY, ANTWERP, BELGIUM.
Keyword: p.argentipes, sand fly; kala-azar, visceral leishmaniasis; housing condition, vector control
Abstract: Introduction: Visceral leishmaniasis or kala-azar is a vector borne chronic infectious disease transmitted by female Phlebotomine argentipes sand flies. Vector control using Indoor Residual Spraying of houses and cattle sheds in the VL affected areas is one of the strategies to combat this disease. We conducted an entomological survey in Muzaffarpur district in Bihar state of India to investigate the role of housing structure, inside and peripheral conditions and socioeconomic status on the abundance of P. argentipes. Method: CDC light traps were installed to capture sand flies in 500 households of 50 villages for 6 nights, one night in each of the three seasons of summer, rainy and winter in two consecutive years. Insects thus collected were morphologically identified and pooled as per their species. Information regarding housing conditions i.e. main materials used in floor, walls and roof, dampness of floor, penetration of daylight, cross ventilation, presence of window, cooking stoves and keeping animals inside was collected using a specifically designed questionnaire. Data regarding immediate surroundings, recent case history and ownership of tangible assets were also collected for each of the households. Results: Overall 49126 sand flies could be collected of which 22430 (45.66%) were P.argentipes. Highest density was observed in rainy seasons while lowest in summer. We found statistically significant associations between P.argentipes density and type of floor, roof and walls. Significantly higher numbers of P.argentipes were found in the thatched houses as compared to brick houses. Cross ventilation, keeping animal inside the room during nights and household assets were also significantly associated with the P.argentipes density. Sun light penetration, kindling cocking stove inside the room, piles of animal dung, garbage collection and temporary water bodies in the immediate neighborhood of the households were not significant at all. Highest density of P.argentipes was observed in the houses of poorest while the richest had the lowest and this difference was significant. Conclusion: Improving housing conditions and living standard helps reducing the P.argentipes density in the households. Vector control program should focus more on the villages of poor housing facilities.
- CLINICAL LEISHMANIASIS [429] O 013 - CLINICAL - LABORATORY SIGNIFICANCES OF SEVERTY LEISCHMANIASIS ANDRIC, B.1; TERZIC, D.1; DUPANOVIC, B.1; DRAGAS, S.1; ICEVIC, M.2 1.CLINIC FOR INFECTIOUS DISEASES, CLINIC CENTER OF MONTENEGRO, PODGORICA, MONTENEGRO, BOSNIA AND HERZEGOVINA; 2.HOFFMAN LA ROCHE LTD., PODGORICA, MONTENEGRO, BOSNIA AND HERZEGOVINA.
Keyword:leishmaniasis ; increase severity; diagnostic difficulties
Abstract: Leishmaniasis to belong of the group of parasitic communicable zoonoses, caused by members of protozoa leishmania species (spp). The infected phlebotomine sand fly carry the parasites which cause the different forms of disease. Visceral leischmaniasis (VL) cases increase around the World, with approximately 500.000 cases per year. Retrospective review of records for documented cases of VL between the period 1992 to 2012 in Montenegro present 81 diagnostic cases (with four deaths cases). The children population participate with 35 cases, the adults with 42 cases. Examinations based on epidemiological, clinical, chematological, patohystological and serological investigations. Infection can be sub-clinically or clinically manifested with acute, sub-acute and chronic course. Incubation in clinically manifested infections ranges from several weeks to several months. In our study the prevalence of general infective syndrome (high febricity, exhaustion) in all cases (100%), enlarged of spleen in 38 cases (%), and liver in 36 (%) cases, anemia in 39 (%) cases, pancytopenia in 22 (%) cases, increased activity of serum transaminases in 27 ( %) cases. The diagnosis was etiologically confirmed through test of agglutination serologically and analysis, bone marrow biopsy by direct microscopy of serial sections colored with the Giemsa s stain, Reticulin (Gordon and Sweet method), PAS method, and by immune-biochemical methods. In Montenegro (in humans and dogs), presented two types of leishmania: l. donovani and l.infantum. As to therapy treatment, the common treating with pentavalent antimony: glucantime was relatively satisfactory over long period of years. During 2008 the first case that did not respond to usual therapy treatment with glucantime was registered. Montenegro is an endemic area for VL. Natural condition and geographical position (Mediteranean area) allow of disease existance. Disbalance of Eco-system, increase of vector density and reservoirs of leishmania spp., to challenge the expansion of the primary endemic focus, and number of infected cases. Also include co-infections among different species of leishmania, as well as leishmania with other microorganisms from the group of vector borne diseases in common endemic areas. Hypothesis of long-term persistence of live leishmania after the infection, classifies them into a group of significant opportunistic agents. - CLINICAL LEISHMANIASIS [395] O 014 - LEISHMANIA INFANTUM TEGUMENTARY LEISHMANIASIS IN SOUTHERN FRANCE. RETROSPECTIVE STUDY 1998-2009. DEDET, J.1; PRATLONG, F.2; MARTY, P.3; FARAUT, F.4; POMARES, C.3; FAUCHER, B.4; BASTIEN, P.1 1.UNIVERSITÉ MONTPELLIER 1, MONTPELLIER, FRANCE; 2.CHRU DE MONTPELLIER, MONTPELLIER, FRANCE; 3.CHU DE NICE, NICE, FRANCE; 4.UNIVERSITÉ AIX-MARSEILLE, MARSEILLE, FRANCE.
Keyword:cutaneous leishmaniasis; mucosal leishmaniasis; southern france
Abstract: (1) Département de Parasitologie-Mycologie, CHRU de Montpellier et Université Montpellier 1, Centre National de référence des leishmanioses, UMR MIVEGEC (CNRS 5290, IRD 224, UM1, UM2), France. (2) Laboratoire de Parasitologie-Mycologie, CHU de Nice, Faculté de Médecine, INSERM U1065, Nice, France
(3) Laboratoire de Parasitologie-Mycologie, UMR MD3, Aix-Marseille University, France
Leishmaniasis is endemic in Southern France, where the single species Leishmania infantum is present. We present a retrospective study of the tegumentary cases reported during 12 years (1998-2009) at the French National Reference Centre for leishmaniasis FNRCL, with special reference to epidémiological, clinical and therapeutical aspects. Visceral leishmaniasis (VL) is the predominant clinical outcome (243 cases out of 280 reported autochthonous cases, 86.8 %), while the number of cutaneous cases (CL) is reduced (n=28, 10.0 %), and mucosal (ML) exceptional (n=9, 3.2 %) CL and ML cases were from the main South of France foci : Pyrénées-Orientales, Cévennes, Provence, Alpes-Maritimes. Sporadic cases of CL were found in all foci, particularly in Cévennes and Alpes-Maritimes. Clinical polymorphism was high and cases probably underestimated. Treatment was diverse, including systemic and local treatment, with the various products available. ML cases were exceptionnal, with limited lesions, always treated by systemic way, either by meglumine antimoniate or liposomal amphotericin B.. Since 2006, a specific structure for therapeutic telephonic advice was established at the FNRCL.. - OTHER SPECIAL TOPICS [746] O 015 - ASYMPTOMATIC INFECTIONS, THE EXPERIENCE FROM THE INDIAN SUBCONTINENT HASKER, E.1; KANSAL, S.2; MALAVIYA, P.2; GIDWANI, K.; SINGH, R.P.2; SINGH, O.P.2; CHOURASIA, A.2; SINGH, A.K.2; SHANKAR, R.2; OSTYN, B.1; WILSON, M.E.3; KHANAL, B.4; RIJAL, S.4; BOELAERT, M.1; SUNDAR, S.2 1.ITM, ANTWERP, BELGIUM; 2.BHU, VARANASI, INDIA; 3.UNIVERSITY OF IOWA, IOWA, UNITED STATES; 4.BPKIHS, DHARAN, NEPAL.
Keyword:subclinical; infection; serology
Abstract: INTRODUCTION: Of all persons infected with the parasites causing visceral leishmaniasis (VL) usually only 10-25% progress to clinical disease. We explored datasets from three different VL endemic study populations in India and Nepal to describe patterns of markers of Leishmania donovani infection and clinical VL. Our total study population was made up of 32,564 individuals. METHODS: In each of the three study populations house to house surveys were conducted during which blood samples on filter paper were collected from all consenting individuals aged 2 years and above on at least two occasions. All baseline samples were tested for antileishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA, follow-up samples were tested with both assays in two studies and only with DAT in one study. Results from successive surveys were used to identify sero-convertors among those with negative serology at
baseline. Data collected during the surveys included information on episodes of clinical VL among study participants. RESULTS: Initial DAT sero-prevalence ranged from 6.2 to 14.8%, rK39 seroprevalence ranged from 5.9 to 16.5%. DAT titers followed a bimodal distribution, rK39 titers were uni-modally distributed. Agreement between DAT and rK39 was limited with kappa values ranging from 0.30 to 0.46. In all three populations the probability of being DAT positive increased with age. DAT sero-conversions were observed in 2.6 to 4.6% of initially sero-negatives; in all three study populations proportions of sero-convertors increased steadily with age. Clinical VL occurred mainly among children and young adults (median ages 13-19 years). Within a one year interval 24 - 33% of initial DAT positives and 59% of initial rK39 positives had reverted back to seronegative; in the study with a 6-month interval between surveys 20% of DAT positives and 17% of rK39 positives had reconverted. DISCUSSION: Infection with L. donovani is assumed to be permanent but serological markers revert back to negative. Though clinical VL is more common at younger ages, we observed a steady increase with age in the frequency of sero-positivity and of sero-conversion. Individuals in endemic areas apparently experience repeated episodes of sero-positivity. This can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. Either mechanism can lead to misclassification of infected subjects. - CLINICAL LEISHMANIASIS [815] O 016 - VISCERAL LEISHMANIASIS IN SOLID ORGAN TRANSPLANTATION (SOT) IN THE CONTEXT OF AN OUTBREAK IN SOUTHWEST MADRID, SPAIN CARRILLO, E.1; CARRASCO, N.2; MEDRANO, F.2; SALTO, E.3; SAN MARTIN, J.V.4; ALVAR, J.5; AGUADO, J.M.2; MORENO, J.1 1.WHO COLLABORATING CENTRE FOR LEISHMANIASIS, C NACIONAL MICROBIOLOGIA, INSTITUTO DE SALUD CARLOS III, MADRID, SPAIN; 2.UNIDAD DE ENFERMEDADES INFECCIOSAS, HOSPITAL UNIVERSITARIO 12 DE OCTUBRE, UNIVERSIDAD COMPLUTENSE, MADRID, SPAIN; 3.SERVICIO DE MICROBIOLOGÍA, HOSPITAL UNIVERSITARIO 12 DE OCTUBRE, UNIVERSIDAD COMPLUTENSE MADRID, MADRID, SPAIN; 4.SERVICIO DE MEDICINA INTERNA, HOSPITAL DE FUENLABRADA, MADRID, SPAIN; 5.DNDI (FORMELY WHO-NTD), GENEVA, SWITZERLAND.
Keyword:solid organ trasplant; visceral leishmaniasis; cryptic
Abstract: The current outbreak of leishmaniasis in the southwest of Madrid (Fuenlabrada town) has reported more than 400 clinical cases since late 2009, representing a prevalence of leishmaniasis of 0,2% in Fuenlabrada town. Spain is one of the countries with the higher number of organ donation and transplantations over the world. In inhabitants of Fuenlabrada town, 34 SOT were performed during this period, and 4 of them developed visceral leishmaniasis (prevalence of 12%). The high susceptibility of this immunodepressed population suggest the vigilance of more than 80 transplanted recipients living in Fuenlabrada town with an increased risk to get infected by Leishmania infantum. The aim of this study is to know the predisposition of transplanted individuals to suffer leishmaniasis in a high transmission area. Their close surveillance will also allow provide the prevalence of cryptic leishmaniasis in this population. Up to now, we have characterized the humoral and cellular immune response against Leishmania in 38 transplant recipients from Fuenlabrada town, without a previous reported episode of leishmaniasis. 9 of them had a patent total soluble Leishmania antigen (TSLA)lymphoproliferative response (23.68%). Cytokine levels from the supernatants were compared to those from whole-blood cytokine release assay, proposed as a complementary exposure test. Culture in NNN medium and PCR were negative in blood of transplanted recipients. As a remarkable finding, one of the transplant individuals with a strong specific cellular immune response also presented anti-Leishmania antibodies, assessed by IFAT, rK39 dipstick and
TSLA- IgG ELISA. The follow up of these transplant recipients, as a measure of control of this population at risk to suffer leishmaniasis due to their immunosuppressed status, will provide guidelines to handle how to proceed with these individuals in a high transmission area. Funded by World Health Organization – APW 2012/271093-0 - CLINICAL LEISHMANIASIS [1214] O 017 - NUTRITIONAL ASSESSMENT IN PATIENTS WITH VISCERAL LEISHMANIASIS. COSTA, C.H.N.1; ROCHA, R.L.2; COSTA, D.L.1 1.FEDERAL UNIVERSITY OF PIAUÍ, TERESINA, PI, BRAZIL; 2.FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:malnutrition; nutritional assessment; wasting
Abstract: Backgound: Protein-energy malnutrition (PEM) and visceral leishmaniasis (VL) affect millions of people worldwide. It has been accepted that PEM is a risk factor for acquiring or VL. Moreover, VL may aggravate a preexisting PEM. Patients and Methods: A prospective, hospital-based study was conducted in the northeast of Brazil, from September 2005 to August 2008. The nutritional status of 883 patients with VL was assessed. Weight and height were measured at the time of admission. When information about the amount of weight loss during the illness was available, the percentage of body weight lost was calculated. Nutritional assessment was performed by the statistical program Epi Info and other analysis were performed by Stata/SE. Results: Weight loss during the period of illness was reported in 639 (72.4%) patients, and 542 (61.3%) of them were able to quantify this loss. The average weight loss was 8.9% of body mass. This loss was significantly more pronounced among patients with lethal outcome than in survivors (p 98%) were significantly delayed in the onset of apoptosis following HNP-1 treatment. This could be envisaged to benefit the neutrophil defense against parasite invasion. Moreover, there was a noteworthy increase in necrotic to apoptotic population ratio in HNP-1 and/or CpG–treated neutrophils in comparison with untreated cells; an advantageous for immune system to evade silently and to reduce parasite chance to exploit apoptotic neutrophils for macrophage infection. In vitro human cytokine assay showed a considerable increase in TNF-α and decrease of TGF-β in HNP-1 (with/without CpG motif)-treated cell cultures, a response that should potentiate the immune system against parasite invasion. In addition, we investigated the effect of HNP-1 on parasite propagation and rate of infectivity in vitro using real-time PCR. Our results showed that the infectivity rate was significantly reduced in treated cells (20-80% reduction). The obtained results show promising features for AMPs as a new class of anti-leishmanial agents and neutrophils as the first effector cells to reduce parasite infection. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [120] O 240 - MONOCYTE-DERIVED TNF AND METALLOPROTEINASE 9 IN PATIENTS WITH CUTANEOUS LEISHMANIASIS PASSOS, S.T.1; CAMPOS, T.2; COSTA, R.2; QUEIROZ, A.2; MOSSER, D.3; SCOTT, P.4; CARVALHO, E.2; CARVALHO, L.2 1.FEDERAL UNIVERSITY OF BAHIA, SALVADOR, BA, BRAZIL; 2.UFBA, SALVADOR, BA, BRAZIL; 3.UNIVERSITY OF MARYLAND, MARYLAND, UNITED STATES; 4.UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, UNITED STATES.
Keyword:cutaneous leishmaniasis; monocytes; matrix metalloproteinases
Abstract: Cutaneous leishmaniasis caused by Leishmania braziliensis is characterized by the presence of one or more ulcerated lesions of raised edges. CD4 + and CD8 + serve as source producing cytokines that activate macrophages to destroy the parasites. Circulating monocytes are a heterogeneous population subdivided into classical monocytes (CD14 + CD16), intermediate monocytes (CD14 + CD16 +) and non-classical monocytes (CD14-CD16 +), and are known to migrate to inflammatory sites and secrete cytokines. TNF can mediate the pathology of CL through various mechanisms including, induction of nitric oxide (NO), increased cytotoxicity and expression of matrix metalloproteinases (MMPs). MMP-9 is a zinc-dependent enzyme which degrades type IV collagen, a component of basal membrane, and is controlled by TIMP-1 (tissue inhibitor of metalloproteinase 1). Recently, it was demonstrated that infection of macrophages by L. braziliensis increases the expression of MMP-9. Although the factors that induce the breakdown of the basal membrane leading to the development of ulcer are unknown, it is likely that MMP-9 contributes to tissue injury in the CL. Our aim was to investigate the contribution of sub-populations of monocytes to production of MMP-9 and their participation in the development of tissue injury in infection by L. braziliensis. The study design was crosssectional study with 28 patients with CL, 12 with early CL and 20 healthy controls. Mononuclear cells were obtained for ex-vivo labeling of sub-populations of monocytes and
MMP-9, and the frequency determined by flow cytometry. Culture was performed for 72 hours, stimulating the cells with SLA, levels of MMP-9 and TIMP-1 in supernatants were determined by ELISA. Our results showed that monocytes were major cells producing MMP-9 and all three sub-populations of monocytes from patients with early CL and CL more expressed MMP-9, compared with those in healthy individuals. In patients with CL non-classical monocytes producer was the main source of MMP-9. Elevated levels of MMP-9 and lower levels of TIMP1 were found in culture supernatants from PBMC of patients with cutaneous leishmaniasis. The contribution of TNF to MMP-9 production was determined in presence or absence of recombinant TNF for 24 hours. The presence of TNF induced MMP-9 secretion as determined by ELISA. In conclusion, high levels of TNF in patients with cutaneous leishmaniasis contribute to MMP-9 production by monocytes. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [83] O 241 - HUMAN MONOCYTES CONTROL LEISHMANIA BRAZILIENSIS BY THE PRODUCTION OF REACTIVE OXYGEN SPECIES NOVAIS, F.1; NYUGEN, B.1; CARVALHO, L.P.2; BEITING, D.P.1; CARVALHO, E.M.2; SCOTT, P.1 1.UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, UNITED STATES; 2.UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL.
Keyword:reactive oxygen species; human monocytes; leishmania braziliensis
Abstract: Leishmania is controlled by the development of Th1 responses that drive IFN-γ-dependent activation of phagocytes and parasite killing through nitric oxide production. This pathway is well established in mouse models, however it is less clear if nitric oxide controls Leishmania parasites in humans. We performed microarray analysis in lesions from L. braziliensis patients and found no changes in inducible nitric oxide synthase expression in comparison to normal skin. These results suggest that other mechanisms of parasite control are required in humans. We found that mice deficient in the generation of reactive oxygen species (ROS) were more susceptible to L. braziliensis than control mice. Hence, we hypothesized that L. braziliensis control in human cells might be dependent on the generation of reactive oxygen species. To test that, we cultured human monocytes with L. braziliensis and assessed the production of ROS and parasite survival. We found that L. braziliensis stimulated a respiratory burst in monocytes, and importantly that the parasites grew substantially better in monocytes in the presence of a ROS inhibitor. In IFN-γ-activated monocytes control of L. braziliensis was also dependent on ROS and independent of nitric oxide production. Finally, we asked whether human monocyte subsets might differ in their ability to produce ROS. Classical monocytes displayed the greatest respiratory burst, compared with non-classical and intermediate monocytes, suggesting that only one of the monocytes subset is capable of eliminating L. braziliensis through ROS production. Taken together, these results suggest that the respiratory burst is required for both innate and adaptive control of L. braziliensis and provides the insight that different monocyte subsets might play distinct roles upon interaction with Leishmania parasites.
- EPIDEMIOLOGY [665] O 242 - AN EVALUTION OF WHO STANDARD LEISHMANIA STRAINS SHAW, J.J. SAO PAULO UNIVERSITY, SAO PAULO, SP, BRAZIL.
Keyword:strain; standard; evaluation
Abstract: The creation of the WHO standard Leishmania strains was the result of a WHO Expert Committee meeting held in Geneva in 1990 and published as a WHO Technical Report (Wld Hlth Org Techn Rep Ser 793: 1-158). At that time many different strains were being used for different purposes and it was impossible to critically compare results. Also it was becoming evident that there were many species and that new isolates had to be compared to some standard to know what species they belonged to. These standards were distributed to different laboratories throughout the world who in turn re-distributed them to other laboratories. This latter procedure was unfortunate as the committee had recommended that they should only be obtained from reference centers. Different methods of propagation were used which in time may have resulted in selections of different genes. Also in some cases particular strains became contaminated with other Leishmania or even other trypanosomatids resulting in publications that reported erroneous information (Parasit Today 11: 347). Single isolates of the WHO standards of the more important leishmanial pathogens have been selected for sequencing and this data is now available. However, at the moment we do not have a complete list of all the laboratories that hold the WHO standards nor do we have any idea if there is any genetic variation amongst them due to different laboratory procedures, such as passaging and conservation. With these points in mind it is perhaps time that the standards were recalled for rigorous comparisons that not only include standard identification methods such a iso-ennzyme and molecular analyses but biological parameters such as infectivity and pathogenecity. These strains represent benchmarks in leishmaniasis and the initiation of international standards and are reference points for basic identifications. However, they do not preclude the establishment of other standards related to specific biological characters such as pathogenecity or drug susceptibility. But these clearly have to be accepted and tested to confirm their specific qualities and compared in the same tests to the WHO standard for that species. Lastly greater emphasis should be placed on the amastigote stage which unfortunately is hardly ever preserved in crobank collections.
- EPIDEMIOLOGY [283] O 243 - CUTANEOUS LEISHMANIASIS IN PALESTINE: A TWO-DECADE EPIDEMIOLOGICAL SURVEY USING MOLECULAR TOOLS AL-JAWABREH, A.1; DUMAIDI, K.2; NASEREDDIN, A.3; AL-JAWABREH, H.1; ABDEEN, Z.3 1.LEISHMANIASES RESEARCH UNIT, JERICHO, PALESTINA; 2.ARAB AMERICAN UNIVERSITY IN JENIN, FACULTY OF ALLIED MEDICAL SCIENCES, JENIN, PALESTINA; 3.AL-QUDS NUTRITIONAL AND HEALTH RESEARCH INSTITUTE-ALQUDS. FACULTY OF MEDICINE –AL-QUDS UNIVERSITY, JERUSALEM, PALESTINA.
Keyword:cutaneous leishmaniasis; l. major and l. tropica; palestine- jericho
Abstract: Cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major is a common skin infection in the Mediterranean basin including Palestine. Over a period of approximately two decades starting in 1994 and ending in 2012, 1735 suspected cases were referred for laboratory diagnosis using conventional methods, NNN culture and Giemsa-stained smear, and molecularbased techniques targeting ITS1 and RFLP-ing corresponding amplicons. Samples included direct touch smears for microscopy, aspirate for in-vitro culture, tissue and blood on filter papers, unstained smears and stained smears for PCR and RFLP genotyping. The overall prevalence was 53% (913/1735). The patients were collected from 11 Palestinian districts including the endemic focus of Jericho (A’riha). Leishmania spp were isolated from patients in all districts. The genotyping of 423 isolates revealed that L. major (48%) and L. tropica (49%) were equally isolated. Jericho district along the Jordan Valley is a focus for both L. major (56%)
and L. tropica (41%), While the rest of the districts are predominated by L. tropica. No statistically significant difference was found in the distribution of infection between both sexes (P=0.45), regardless of the genotype. About 62%(553/876) of the patients were children below 14 years of age. Based on date of sampling, L. major and L. tropica follow different seasonal patterns. L. major cases are highest in December and January and lowest in April and May. While, L. tropica cases are highest in March and April and lowest in month from July till November. It is concluded that active surveillance and incorporation of battery of laboratory tests including molecular methods for genotyping are crucial for disease control. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [656] O 244 - RECOMBINANT PHLEBOTOMUS PERNICIOSUS SALIVARY PROTEINS AS MARKERS OF HOST EXPOSURE TO SAND FLY BITES MARTÍN-MARTÍN, I.1; DRAHOTA, J.2; SUMOVÁ, P.2; ROHOUŠOVÁ, I.2; JIMENÉZ, M.1; MOLINA, R.1; VOLF, P.2
1.UNIDAD DE ENTOMOLOGÍA MÉDICA, CENTRO NACIONAL DE MICROBIOLOGÍA, INSTITUTO DE SALUD CARLOS III, MADRID, SPAIN; 2.DEPARTMENT OF PARASITOLOGY, FACULTY OF SCIENCE, CHARLES UNIVERSITY IN PRAGUE, PRAGUE, CZECH REPUBLIC.
Keyword:phlebotomus perniciosus; sand fly saliva; recombinant proteins
Abstract: Canine leishmaniasis (CanL) has been traditionally associated in Europe to the Mediterranean basin where the causative agent is Leishmania infantum. Phlebotomus perniciosus acts as the main vector in the Iberian Peninsula. Nowadays CanL is spreading to previously non-endemic areas in Europe. In this scenario, epidemiological tools that help to establish the risk to sand fly exposure would improve control strategies against leishmaniasis. Sand fly bites trigger in hosts a specific humoral immune response against vector saliva. Therefore, measuring antibodies antisand fly saliva can be used to assess the exposure of host to sand fly bites and thus the risk of Leishmania transmission. Working with the salivary extract show several drawbacks such as the variability of salivary gland contents according to physiological factors and therefore, recombinant salivary proteins (rSP) that could replace the salivary extract are being sought. In this work, several recombinant salivary proteins from P. perniciosus obtained under different expression and purification conditions were tested as potential markers of sand fly exposure by ELISA and Western lot (WB). Experiments with mice sera showed that both apyrases (SP01 and SP01B), yellow protein (SP03B), and Para25-like (SP08) were recognized by sera of mice immunized with sand fly saliva. However, the D7-related protein (SP04) and the antigen 5 (SP07) did not discriminate between sera of exposed and non exposed mice either by ELISA or WB. When testing the rSP with canine sera of dogs experimentally exposed to sand fly bites in ELISA, best results were found with several recombinant salivary proteins SP01, SP01B and SP03B. A significant positive correlation between these rSP and the salivary extract homogenate was found as determined by Spearman test (SP01 r=0.8493, p 0.05). The sensitivity and specificity of the conventional PCR were calculated based on the parasitological test using bone marrow cultures. The PCR associated with bone marrow samples presented the highest sensitivity ( 93,8%) followed by nasal swab samples (87,5%). The specificities were 54,2% and 50% respectively. The parasite burden in nasal swab was statistically equivalent to the parasitism in conjunctival swab (p>0.05) and lower than the parasite load in bone marrow and skin biopsy (p70%) reduction in parasite loads in the liver and spleen. Furthermore, liposomal pentalinosterol treated mice mount a mixed Th1/Th2 response but produce significantly more IFN-γ compared to controls treated with empty liposomes. Taken together, these data indicate that pentalinosterol could be novel lead molecule for developing new drugs against leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [41] O 309 - INHIBITORS OF LEISHMANIAL DIPEPTIDYLCARBOXYPEPTIDASE: AN APPROACH FOR DRUG DISCOVERY GOYAL, N.G. CENTRAL DRUG RESEARCH INSTITUTE, LUCKNOW, INDIA.
Keyword:dipeptidylcarboxypeptidase; inhibitor; leishmanicidal activity
Abstract: Current treatment of leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations such as high cost, toxicity and lack of efficacy in endemic regions. Therefore, development of new, effective and affordable anti-leishmanial drugs is a global health priority. Dipeptidylcarboxypeptidase of Leishmania donovani (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase, has been characterized as novel drug target for antileishmanial drug discovery. By virtual screening of Institute’s chemical library of 15,452 compounds against a 3D model of LdDCP, four compounds, belonging to two chemical classes, were identified as the selective inhibitor of parasite enzyme. These compounds also inhibited multiplication of both promastigotes and amastigotes under in vitro condition. One of these compounds also exhibited promising in vivo anti-leishmanial activity in hamster model. The
data suggests that these compounds provide leads to be optimized into candidates to treat these protozoan infections. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [313] O 310 - A NOVEL ALKYL PHOSPHOCHOLINE-DINITROANILINE HYBRID MOLECULE EXHIBITS BIOLOGICAL ACTIVITY IN VITRO AGAINST LEISHMANIA AMAZONENSIS. GODINHO, J.L.P.1; GEORGIKOPOULOU, K.2; CALOGEROPOULOU, T.2; DE SOUZA, W.1; RODRIGUES, J.C.F.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.INSTITUTE OF ORGANIC AND PHARMACEUTICAL CHEMISTRY, ATHENS, GREECE., GREECE.
Keyword:alkyl phosphocholine-dinitroaniline hybrid; leishmania amazonensis; cellular targets
Abstract: Leishmaniasis is one of the most important neglected tropical diseases caused by parasites of the Leishmania genus. The current chemotherapy is based on pentavalent antimonials, miltefosine, amphotericin B and pentamidine. However, there is an urgent need for safer and more efficacious drugs. An interesting approach in drug development is the combination therapy using drugs with known activity against the parasites. Both, trifluralin, a dinitroaniline herbicide, and miltefosine have activity against protozoan parasites, but with high IC50 and cytotoxicity for host cells. In this work, we investigated the effects of TC95 on Leishmania amazonensis. TC95 is a hybrid compound that combines trifluralin and miltefosine in only one molecular scaffold. The antiproliferative effects of TC95 against promastigotes were dosedependent, with a total inhibition at concentrations of 4 and 5 μM. Against intracellular amastigotes, the effect was also dose-dependent after 24 h of treatment, however the effect was more pronounced after 48 h of treatment for all concentrations tested [1, 5 and 10 μM]. The IC50 values obtained after 48 h of treatment was 2.6 μM and 1.2 μM for promastigotes and intracellular amastigotes, respectively. Cytotoxicity assays against murine macrophages revealed that the CC50 was 60 μM, thus presenting a selective index of 24x. From 12 h until 72 h of treatment, promastigotes displayed profound alteration in the shape appearing rounded and swollen, with alterations in the cell cycle and in the cytoskeleton constituted mainly by microtubules. Transmission electron microscopy revealed that the mitochondrion is the main organelle affected, presenting an intense swelling with loss of the matrix content and disorganization of the mitochondrial membranes. Sometimes, lysis of the mitochondrion was also observed. Furthermore, alterations in the flagellar membrane, Golgi complex, increase in the number of lipid bodies and appearance of structures typically found in autophagic processes were also observed. Against intracellular amastigotes, TC95 also induced significant alterations such as: mitochondrial swelling with vesiculation of the mitochondrial membranes and alterations in the kinetoplast; significant dilation of the trans-Golgi network; presence of lipid bodies in the amastigotes and inside the macrophages; and changes in the flagellar structure. Taken together, these results indicate that TC95 is a promising compound against Leishmania sp. Financial supported by CNPq, CAPES, and FAPERJ.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [450] O 311 - AMPHIPHILIC ANTIMONY COMPLEXES: A SUCCESSFUL STRATEGY FOR ORAL TREATMENT OF EXPERIMENTAL VISCERAL LEISHMANIASIS
FERNANDES, F.R.; FERREIRA, W.; ALBANEZ, M.; KATO, K.; RAMOS, G.S.; MELLO, M.N.; DEMICHELI, C.; FREZARD, F. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:amphiphilic antimony complexes; oral treatment ; visceral leishmaniasis
Abstract: Pentavalent antimonial drugs, in spite of their severe side effects, have been used to date in the treatment of all forms of leishmaniasis. Due to their low oral bioavailability, these compounds must be parenterally administered, compromising patient compliance. The objetives of this work were to prepare amphiphilic antimony complexes from alkylmethylglucamide (L8 and L10, hydrophobic chain of 8 and 10 carbons, respectively) and to evaluate their potential for oral treatment of visceral leishmaniasis (VL). The reaction of Sb(V) with L8 and L10 produced 1:3 Sb-ligand complexes, as evidenced by NMR and ESI-MS analyses, and stabilized the formation of nanoassemblies in water. According to pharmacokinetic studies in mice by the oral route, SbL8 complex at 200 mg Sb/kg showed higher and more sustained Sb levels in the serum and greater affinity for the liver, when compared to conventional Glucantime® at the same Sb dose. SbL8 complex showed a peak concentration in the liver before that in the serum, suggesting an initial absorption to the liver followed by a gradual release of Sb to the circulation. The efficacies of SbL8 and SbL10 were evaluated by the oral route in BALB/c mice infected with Leishmania infantum after daily dose of 200 mg Sb/kg for 30 days. Quantification of the parasite load in the liver and spleen by the limiting dilution assay showed significant parasite suppression in the liver, in relation to control untreated group, and this difference was also observed in the spleen of animals treated with SbL10. The group treated intraperitoneally with Glucantime® (80 mg Sb/kg/day) also showed significant parasite supression. As expected, there was no significant reduction of the parasitic load in the group treated orally with Glucantime® at 200 mg Sb/kg/day for 30 days. In conclusion, amphiphilic antimony(V) complexes emerge as an innovative and promising strategy for the oral treatment of VL. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [750] O 312 - ORYZALIN NANOFORMULATIONS AS ANTILEISHMANIAL AGENTS: IN VITRO AND IN VIVO EVALUATION CRUZ, M.E.M.1; LOPES, R.M.1; ELEUTÉRIO, C.1; CARVALHEIRO, M.1; CORVO, M.L.1; GONÇALVES, L.M.1; GASPAR, M.M.1; SCOULICA, E.2; ALMEIDA, A.J.1 1.FACULDADE DE FARMÁCIA, LISBOA, PORTUGAL; 2.SCHOOL OF MEDICINE, CRETE, GREECE.
Keyword:oryzalin; nanoformulations; leishmaniasis
Abstract: Dinitroanilines, such as Oryzalin (ORZ), are tubulin-binding agents that display antimitotic activity on Leishmania sp [1]. In spite of its potentiality, the therapeutic use is limited by low water solubility associated with rapid clearance in vivo. These disadvantages can be overcome by ORZ incorporation in lipidic nanoformulations, namely liposomes and solid lipid nanoparticles (LNP), that act simultaneously as drug solvent and as carriers delivering ORZ to the sites of Leishmania infection potentiating ORZ antileishmanial activity. Liposomal and LNP formulations containing ORZ were optimized by selection of the appropriated preparation method, lipidic components and other experimental conditions in order to obtain formulations suitable and safe for parenteral administration. Both systems efficiently incorporated ORZ allowing the increase of concentration in aqueous suspensions at least 150 times without the need of toxic solvents. The developed formulations revealed to be stable throughout freeze-drying and moist-heath sterilization without significant variations on
physicochemical properties namely on ORZ retention. These formulations presented sizes in the nano range (100-200 nm) [2,3]. The incorporation of ORZ in both liposomes and LNP remarkably decreased hemolytic activity on red blood cells and drug cytotoxicity on THP1 cell line as compared with free drug [2,3]. Intracellular activity studies in L. infatum THP1 infected cells showed that ORZ retains its activity even after incorporation in these nanoformulations. Biodistribution studies of dual labeled ORZ liposomal formulations,(3H-LIP-14C-ORZ) demonstrated the efficacy of liposomes to passively target ORZ to the main leishmanial infected organs (liver and spleen) [2]. The evaluation of the therapeutic activity in L. infantum animal model demonstrated the superiority of ORZ liposomal and LNP formulations on the reduction of the parasite load in liver and spleen as compared to the control group (89 and 84 % for liposomal formulations and 84 and 91% for LNP formulations, respectively), and similar to the standard drug Glucantime. This work was supported by FCT (PTDC/CVT/098290/2008 and PEst-OE/SAU/UI4013/2011). R. Lopes was recipient of a FCT grant SFRH/BD/44218/2008. [1] Morrissette, NS et. al., Mol Biol Cell (2010) 15(4), 1960-8 [2] Lopes R et al., Eur J Pharm Biopharm. (2012) 82(2), 281-90 [3] Lopes R et al., Eur J Pharm Sci. (2012);45(4):442-50
- CONTROL PROGRAMS [23] O 313 - VISCERAL LEISHMANIASIS CONTROL: DIFFICULTIES AND NEW APPROACHES BOELAERT, M. INSTITUTE OF TROPICAL MEDICINE, ANTWERPEN, BELGIUM.
Keyword:visceral leishmaniasis; control methods; methodological challenges
Abstract: Current Visceral Leishmaniasis (VL) control depends on a combination of vector control, case management and reservoir control if relevant. The evidence base in support of each of these strategies is largely empirical or based on pilot studies, with few large-scale controlled trials establishing impact on transmission. A recent systematic review of the evidence on VL control methods in Latin-America showed a persisting evidence gap. Innovation in vector control approaches has so far concentrated on insecticide-treated materials, and more recently on topical or systemic treatment of animals. Active case detection approaches have been advocated and are now piloted within the VL elimination initiative in the Asian subcontinent. The impact on transmission of recently registered dog vaccines still needs to be established in areas with zoonotic transmission. We will discuss the methodological challenges posed by this type of impact studies as an introduction to this session on state-of-the art VL control methods. - CLINICAL LEISHMANIASIS [140] O 314 - GLOBALLY FIRST TIME INTRODUCTION OF LLIN (LONG LASTING INSECTICIDE TREATED NET) ACCORDING TO WHO GUIDELINE, AND ITS IMPACT
IN KALA-AZAR (VISCERAL LEISHMANIASIS ) ELIMINATION IN HYPER-ENDEMIC SUB-DISTRICTS OF BANGLADESH. CHOUDHURY, T.A. VISCERAL LEISHMANIASIS (KALA-AZAR) ELIMINATION PROGRAM, DIRECTORATE GENERAL OF HEALTH SERVICES, MINI, DHAKA, BANGLADESH.
Keyword:long lasting insecticide treated net; vl elimination program; hyper endemic subdistrict
Abstract: responsible for overall management of each booth. 3.) One LLIN for each patient and extra one for his/her family members were distributed. If there was any family having more than two patients in that case extra one LLIN has been given for each patient. Follow up at LLINs: LLIN-use were followed up by the respective healthy assistant / AHI as per their monthly field tour. This follow up was monitored by the HI & SI at the union level. At the Sub-district level Sub-district Health & Family Planning Officer & at the district level Civil Surgeon was responsible for monitoring the whole distribution & follow up of LLIN. At the central level Director, Disease Control & Line Director, Control of Communicable Disease, and Assistant Director, Deputy Director , Deputy Program Manager-Kala-azar, Consultant (Kala-azar) and Medical Officer(Kala-azar) monitored and supervised time to time to ensure proper LLINs distribution and its follow-up. BCC activities before & after LLINs distribution: BCC materials like leaflet, poster, banner, sticker etc has been developed before distribution of LLIN. All BCC materials has been distributed & displayed 1-2 days before distribution of LLIN. Milking at the community level were done before LLINs distribution campaign at the union/village level. Proper Inter Personal Communication before and after organizing LLINs distribution was done Teach People How to Use and Care for Nets: Handle the nets gently to avoid tearing them Fold/tie up the net during the day to avoid damage Regularly inspect the net for holes; repair holes, if found LLINs last for three to five years or 20 washes, but may expire sooner if washed too often Wash only when very dirty, no more than a few times a year Wash nets with gentle soap, NOT detergent
Dry nets in the shade—no sun because it will destroy the chemical Keep LLINs away from smoke, fire, direct sunlight Deliverables: • Globally practiced for first time, as a tool for Integrated Vector Management (IVM) for Visceral Leishmaniasis Elimination Program, LLIN distribution was successfully implemented and can be practiced in other countries. • To reduce the transmission of Kala-azar from reservoir to Vector, LLIN distribution can play a vital role for PKDL patients. • Lessons learned that LLIN distribution among KA/KDL cases, with prior campaign for community participation can act as Catch-up strategy for new case identification.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [69] O 315 - CAN ELIMINATION PROGRAMME BE ACHIEVED WITH MILTEFOSINE IN THE INDIAN SUBCONTINENT SUNDAR, S.1; DUJARDIN, J.2; BOELART, M.3; OSTYN, B.4; RAI, M.5; SINGH, A.6; PRAJAPATI, V.K.7; SINGH, A.K.8; CHAKRAVARTY, J.9 1.INSTITUTE OF MEDICAL SCIENCES, VARANASI, INDIA; 2.INSTITITE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 3.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 4.; 5.; 6.; 7.; 8.; 9..
Keyword:visceral leishmaniasis; miltefosine; drug resistance
Abstract: Miltefosine is the only oral drug available for treatment of Indian Visceral Leishmaniasis (VL) which was shown to have an efficacy of 94% in a phase 3 trial in Indian subcontinent. The drug has been chosen to be the only primary drug for the elimination of VL from the Indian subcontinent. Its unrestricted use has raised concern about its continued effectiveness. Reports of its efficacy from Nepal and Bangladesh are not favorable This study evaluates the efficacy and safety of miltefosine for the treatment of VL after a decade of use in India. In this study, which was an open-label non-comparative trial, 567 patients received oral Miltefosine (50 mg for those weighing 25 kg, and 2.5 mg per kg for children < 12years, daily for 28 days) in a directly observed manner. Patients were followed up for six months to see the response to therapy. At the end of treatment the initial cure rate was 97.5% (intention to treat) and at six month the final cure rate was 90.3%. Overall death rate was 0.9% (5/567) and two deaths were drugtoxicity related. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 (1.5%) patients due to the adverse events of the drug. To conclude, as compared to phase 3 trial which lead to registration of the drug a decade ago, there is a substantial increase in the failure rate of oral miltefosine for treatment of VL in India.
- OPERATIONAL HEALTH [547] O 316 - VILLAGE HEALTH WORKERS IN BIHAR, INDIA: AN UNTAPPED RESOURCE IN THE STRUGGLE AGAINST KALA-AZAR MALAVIYA, P.1; HASKER, E.2; SINGH, R.P.1; GEERTRUYDEN, J.V.3; BOELAERT, M.2; SUNDAR, S.1 1.BANARAS HINDU UNIVERSITY, VARANASI, INDIA; 2.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 3.ANTWERP UNIVERSITY, ANTWERP, BELGIUM.
Keyword:kap survey, visceral leishmaniasis; drug monitoring, drug effectiveness; public health system, supervised treatment, patient follow-up, auxiliary nurse/midwife, accredited social health activist network
Abstract: Introduction: In 2005 a visceral leishmaniasis (VL) elimination initiative was launched on the Indian subcontinent; important components of early case finding and treatment are entrusted to the primary health care system (PHC). In an earlier study in Bihar, India, we discovered some major shortcomings in implementation, in particular related to monitoring of treatment and treatment outcomes. These shortcomings could be addressed through involvement of village health workers. In the current study we assessed knowledge, attitude and practice of these village health workers in relation to VL. Main objective was to assess the feasibility of their involvement in VL control. Methods: We obtained a list of auxiliary nurses/midwives and accredited social health activists for the highly endemic district of Muzaffarpur. We randomly sampled 100 auxiliary nurses and 100 activists, who were visited in their homes for an interview. Questions were asked on knowledge, attitude and practice related to visceral leishmaniasis and to tuberculosis. The structured questionnaire contained questions exploring training level, professional experience and experience with VL and TB; in addition, there were questions exploring knowledge on presenting signs and symptoms, mode of transmission and diagnostic and treatment procedures of VL and TB, as well as questions exploring willingness to become further involved in VL control. Results: Auxiliary nurses and activists know the presenting symptoms of visceral leishmaniasis. They know how it is diagnosed but they are not aware of the recommended first-line treatment and how the treatment is administered. Many are already involved in tuberculosis control and are very well aware of the treatment modalities of tuberculosis, but very few are involved in the program of visceral leishmaniasis control. They are well organised, have strong links to the primary healthcare system and are ready to get more involved in visceral leishmaniasis control. They need to be given proper training before formal involvement and monetary incentive to compensate for the additional workload. Conclusion: To ensure adequate monitoring of visceral leishmaniasis treatment and treatment outcomes, the control programme urgently needs to consider involving auxiliary nurses and activists. - OTHER SPECIAL TOPICS [922] O 317 - INTERACTIONS AMONG CHICKEN’S COMPLEMENT SYSTEM, LEISHMANIA INFANTUM PROMASTIGOTES AND COMPLEMENT INHIBITORS FROM LUTZOMYIA LONGIPALPIS MENDES-SOUSA, A.F.; QUEIROZ, D.C.; NASCIMENTO, A.A.; ARAÚJO, R.N.; FUJIWARA, R.T.; PEREIRA, M.H.; GONTIJO, N.F. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:complement system; chickens; leishmania
Abstract: Domestic chickens (Gallus gallus) are common blood sources for Lutzomyia longipalpis (Diptera, Psychodidae), the main vector of Leishmania infantum in the New World, and present an important role in the epidemiology of American visceral leishmaniasis. Those animals are abundant in endemic areas and since they kill Leishmania through mechanisms not yet clarified, they could act as zooprophylaxis for the disease. During bloodmeal, the vector inoculates saliva in the host skin, which facilitates the hematophagy as it contains several pharmacologically active molecules. Previous works have demonstrated that L. longipalpis saliva contains inhibitors of the complement system and can protect L. infantum from lysis by human complement system. The objective of this study was to investigate how L. longipalpis interacts with chickens complement system and the role this system has in the parasite clearance by chickens. We evaluated the inhibitory effect of L. longipalpis´ saliva and the intestinal content on both classical and alternative pathways of chickens through hemolytic tests. Through flow cytometry assays, we analyzed the importance of chickens complement system in the elimination of L. infantum promastigotes with different concentrations of sera in presence and in absence of the vector saliva. Results showed that the saliva is not effective to inhibit the classical and the alternative pathways of the avian complement system. On the other hand, the intestinal content inhibited significantly the classical pathway. Chickens serum was very efficient to promote Leishmania death, killing nearly 90% of the parasites even with the less concentrated serum (2%). In the presence of saliva, no significant difference was observed in the parasite death, because the saliva is not capable to inhibit the avian complement. We concluded that the complement system is a very important mechanism through which chickens kill L. infantum and that the saliva of the vector, as it does not inhibit avian complement system, does not protect the parasite. The intestinal inhibitors may be involved in the midgut protection against damage by the membrane attack complex. - RESERVOIRS [534] O 318 - DYNAMICS OF LEISHMANIA INFECTION AND POSSIBLE CORRELATION WITH ANTIBODY RESPONSE AGAINST PHLEBOTOMUS PAPATASI SALIVARY GLAND ANTIGENS IN RHOMBOMYS OPIMUS POPULATION IN CENTRAL IRAN AKHAVAN, A.A.1; YAGHOOBI-ERSHADI, M.R.1; KHAMESIPOUR, A.2; MIRHENDI, H.1; ALIMOHAMMADIAN, M.H.; JEDDI-TEHRANI, M.3; RASSI, Y.1; BATES, P.4; KAMHAWI, S.5; VALENZUELA, J.G.5; VOLF, P.6; MAHMOUDI, A.R.3; GHODS, R.3; ARANDIAN, M.H.7; HOSSEINI, M.1; ABDOLI, H.7; JAFARI, R.7; SHAREGHI, N.7; JALALI-ZAND, N.7; GHANEI, M.7 1.SCHOOL OF PUBLIC HEALTH, TEHRAN UNIVERSITY OF MEDICAL SCIENCES, TEHRAN, IRAN, ISLAMIC REPUBLIC OF; 2.CENTER FOR RESEARCH AND TRAINING IN SKIN DISEASES AND LEPROSY, TEHRAN UNIVERSITY OF MEDICAL SCIENCES, TEHRAN, IRAN, ISLAMIC REPUBLIC OF; 3.MONOCLONAL ANTIBODY RESEARCH CENTER, AVICENNA RESEARCH INSTITUTE, ACECR, TEHRAN, IRAN, ISLAMIC REPUBLIC OF; 4.SCHOOL OF HEALTH AND MEDICINE, LANCASTER UNIVERSITY, LANCASTER, UNITED KINGDOM; 5.NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES, NATIONAL INSTITUTE OF HEALTH, ROCKVILLE, UNITED STATES; 6.FACULTY OF SCIENCE, CHARLES UNIVERSITY, PRAGUE, CZECH REPUBLIC; 7.ISFAHAN STATION, NATIONAL INSTITUTE OF HEALTH RESEARCH, TEHRAN UNIVERSITY OF MEDICAL SCIENCES, ESFAHAN, IRAN, ISLAMIC REPUBLIC OF.
Keyword:rhombomys opimus; leishmania infection, salivary gland antigens; phlebotomus papatasi, iran
Abstract: Zoonotic cutaneous leishmaniasis (ZCL), a neglected tropical disease, is a major public health problem in many rural districts of 17 of 31 provinces in Iran. The disease has been expanding to new foci in recent decades. Rhombomys opimus, the great gerbil, is the main natural reservoir host of the disease and Phlebotomus papatasi is the main vector in central Iran. In the current study, seasonal variation of natural Leishmania infection rates in R. opimus population and
seasonal fluctuations of the antibody (Ab) titer against salivary gland antigens (SGA) of P. papatasi in the reservoir host and possible correlation in central Iran were studied. The investigation was conducted from October 2006 to October 2008 in two endemic foci of ZCL in Esfahan Province, central Iran. Regardless of having any suspected lesions, impression smears were prepared from the ear lobes of the rodents. A Nested PCR assay was designed and used for detection and identification of Leishmania species directly from gerbil's skin samples . ELISA method was used to evaluate anti-SGA Ab titer in wild-caught R. opimus. Out of 111 investigated animals, 2.7% were infected with L. major, 0.9% with L. gerbilli and 35.1% with L. turanica. Mixed natural infections with L. major and L. turanica was found in 16.2% of the rodents, mixed L. major and L. gerbilli in 0.9% and mixed three Leishmania species infections in 1.8% of the gerbils. The highest and the lowest natural L. turanica infection rates were observed in fall (62.5%) and spring (18.2%) respectively. The highest and the lowest mixed infection rates with L. turanica and L. major were also observed in fall (25.8%) and spring (4.6%) correspondingly. A significant (P 0.5, 36 (70.6%) had glomerulonephritis associated with interstitial nephritis. Nineteen dogs (27.9%) had hypertension; all of them with histopathologic evidence and 83.3% with laboratory evidence of glomerular disease. Of the 19 hypertensive dogs, 78.94% (15/19) had membranoproliferative glomerulonephritis associated with glomerular fibrosis, 15.78% (3/19) had membranous glomerulonephritis of intense degree, and one dog presented glomerular fibrosis associated with mild proliferative glomerulonephritis. To the author's knowledge, this is the first study to correlate histological abnormalities with hypertension in dogs with visceral leishmaniasis.
- GENETICS, EVOLUTION AND TAXONOMY [306] P 057 - GENETIC STRUCTURE OF LUTZOMYIA LONGIPALPIS AND LUTZOMYIA CRUZI POPULATIONS IN MATO GROSSO DO SUL, BASED ON MICROSATELLITE MARKERS SANTOS, M.F.C.1; RIBOLLA, P.E.M.2; ALONSO, D.P.2; ANDRADE, J.D.3; CASARIL, A.E.1; FERREIRA, A.M.T.1; FERNANDES, C.E.S.1; BRAZIL, R.P.4; OLIVEIRA, A.G.1 1.UFMS, CAMPO GRANDE, MS, BRAZIL; 2.UNESP, BOTUCATU, SP, BRAZIL; 3.FIOCRUZ - CPRR, BELO HORIZONTE, MG, BRAZIL; 4.FIOCRUZ - IOC, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral leishmaniasis; polymorphisms; sibiling species
Abstract: The main vector of Leishmania (Leishmania) infantum chagasi and therefore essential in the epidemiology of American Visceral Leishmaniasis (AVL), Lutzomyia longipalpis (Diptera: Psychodidae) is the best studied species of sandflies of the neotropical region. Many studies claim that this is in fact a species complex, however there is still no consensus on the number of species that fall into this complex, either on the geographical distribution of sibling groups. In some municipalities of Mato Grosso do Sul there’s evidence of sympatry between Lu. longipalpis and Lu. cruzi so, the aim of this study was to analyze the genetic relationships within Lu. longipalpis complex populations in the state of Mato Grosso do Sul (MS), using as outgroup a population from the Northeast of Brazil. For that purpose we collected 30 individuals (15 females and 15 males) from each locality (Campo Grande, Três Lagoas, Aquidauana, Miranda, Bonito and Corumbá), totalizing 180 sandflies from MS, besides 30 from Estrela de Alagoas, state of Alagoas (AL). The insects were processed through the polymerase chain reaction, using eight microsatellite markers, previously described. The results show that microsatellite
genotyping was efficient to distinguish Lu. longipalpis from Lu. cruzi , either, differentiate Lu. longipalpis collected in Mato Grosso do Sul and those from Estrela de Alagoas. The analyses of the fragments were able to demonstrate that Lu. longipalpis in MS have the same genetic structure, with a minor difference from the sandflies from Três Lagoas. This lead us to believe that there is a single population of Lu. longipalpis in MS, and, the sandflies from Três Lagoas could have derived recently, which means they could be in an early or different stage of speciation. This should be fully investigated in further studies in order to determine which features are implicated in this process. This study provided significant advances in the understanding of the population structure of Lu. longipalpis in MS and, by extension, the neotropical Lu. longipalpis complex itself. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [308] P 058 - MICRORNA EXPRESSION PROFILE IN HUMAN MACROPHAGE IN RESPONSE TO LEISHMANIA MAJOR INFECTION MKANNEZ, G.1; LEMAIRE, J.2; GUSTIN, C.2; GUERFALI, F.Z.1; ATTIA, H.1; SGHAIER, R.M.1; CONSORTIUM, S.1; DELLAGI, K.1; RENARD, P.2; LAOUINI, D.1 1.INSTITUT PASTEUR DE TUNIS, TUNIS-BELVÉDÈRE, TUNISIA; 2.NARILIS-UNIVERSITY OF NAMUR, NAMUR, BELGIUM.
Keyword:microrna, modulation; macrophage, infection; leishmania major
Abstract: Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide noncoding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. We report in this study a comprehensive analysis of miRNA expression profiles in L. majorinfected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24h). We show that expression of 64, out of 365 analyzed miRNAs, were consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibit anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. Our data suggest that alteration in miRNA levels likely play important role in regulating macrophage functions following L. major infection. These rsults could contribute to better understanding the dynamics of gene expression in host cells during leishmaniasis. Work funded by the EU: LSHG-CT-2006-037231. KD and DL were partially supported by an NIH/NIAID/DMID Grant Number 5P50AI074178.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [309] P 059 - TEMPORAL TRANSCRIPTOMIC DYNAMICS OF HUMAN MACROPHAGE RESPONSE TO LEISHMANIA MAJOR INFECTION MKANNEZ, G.1; GHEDIRA, K.1; GUERFALI, F.Z.1; RABHI, I.1; DASKALAKI, A.; TRENTIN, B.2; SGHAIER, R.M.1; ATTIA, H.1; CONSORTIUM, S.1; REGNAULT, B.3; BENKAHLA, A.1; KEL, A.4; PIQUEMAL, D.2; DELLAGI, K.1; LAOUINI, D.1 1.INSTITUT PASTEUR DE TUNIS, TUNIS-BELVÉDÈRE, TUNISIA; 2.SKULD-TECH, MONTPELLIER, FRANCE; 3.INSTITUTE PASTEUR, PARIS, FRANCE; 4.INSTITUTE OF SYSTEMS BIOLOGY, NOVOSIBIRSK, RUSSIA FEDERATION.
Keyword:macrophage, microarray; transcriptome, infection; leishmania major
Abstract: Leishmania (L.) are obligated intracellular protozoan parasites that develop electively in macrophages. These cells that are acting as a safe shelter for the pathogens but also as their ultimate killer, making them the alpha and the omega during leishmaniasis diseases. Macrophages are able to secrete a remarkably diverse set of regulators known to influence the physiological functions and differentiation of neighboring cells to trigger an adaptive immune response of protective Th1-type cells, whereas parasites have developed a wide range of mechanisms to circumvent the host’s immune responses. Most of our understanding of this hostparasite conflict, in the context of macrophage invasion by L. major metacyclic promastigotes, has been gleaned from studies investigating the macrophage responses at late and unique time points after infection. To investigate the dynamics of this duel, we have analyzed the transciptomic profile of monocyte-derived human macrophages at different time points during the first 24h upon in vitro infection using high throughput microarray platform. The gene expression profile of 17,838 genes showed high expression variability between the three human donors at different time points post infection. Cross comparison between the three donors allowed the identification of a common set of expressed genes coding for inflammatory and chemotactic molecules, transcription factors, apoptosis inhibition, glucose synthesis and heme metabolism. The findings presented in this work suggest that transcriptome dynamics of macrophages early during the first 24h post infection enable to identify novel key pathways deregulated upon L. major invasion. Our reported set of expressed genes will be useful in future rounds of data mining and functional analyses. Work funded by the EU: LSHG-CT-2006-037231. KD and DL were partially supported by an NIH/NIAID/DMID Grant Number 5P50AI074178. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [312] P 060 - BONE MARROW PARASITE BURDEN AMONG PATIENTS WITH NEW WORLD KALA-AZAR IS ASSOCIATED WITH DISEASE SEVERITY COSTA, C.H.N.1; SILVA, J.M.1; ZACARIAS, D.A.1; DE FIGUEIRÊDO, L.C.1; SOARES, M.R.A.1; ISHIKAWA, E.A.Y.2; COSTA, D.L.1 1.FEDERAL UNIVERSITY OF PIAUÍ, TERESINA, PI, BRAZIL; 2.FEDERAL UNIVERSITY OF PARÁ, BELÉM, PA, BRAZIL.
Keyword:kala-azar; bone marrow; parasite load
Abstract:
Background: Kala-azar kills 20,000 – 40,000 persons annually, mainly due to a severe form of the disease. The agents, Leishmania donovani and L. infantum, obligatory intracellular protozoa of mononuclear phagocytes found mostly in spleen and bone marrow lead to protracted fever, anemia, wasting, and hepatosplenomegaly. Even after diagnosis, the disease kills around 10% of the patients. Although the pathogenesis of lethal kala-azar is poorly understood, Leishmania specific immunosuppression is a typical feature of kala-azar that may be related to mortality. Methods: We tested if high bone marrow parasite load as measured by quantitative PCR at the hospital admission of 122 patients would predict death and would be associated with clinical symptoms and signs of severity. Results: The data show that parasite load was much higher among deceased patients than among survivors (median 74,975.9 vs. 4,209.3 parasites/106 nucleated host cells). Patients who claimed wasting had higher parasite burden; accordingly, the amount of weight loss and the weight for age z-score were positively correlated with the parasite burden. Also, patients with epistaxis, abdominal pain, edema and jaundice showed more elevated parasite bone marrow parasite load. Conclusion: The study suggests that initial immunological failure to contain parasite replication, besides malnutrition-induced immunodeficiency, may lead to increased antigenic load, which may generate more systemic inflammation, symptoms, signs, and death.
- CLINICAL LEISHMANIASIS [325] P 061 - LEISHMAN: A EUROPEAN CONSORTIUM FOR THE HARMONISATION OF DIAGNOSIS, PARASITE TYPING AND TREATMENT OF PATIENTS WITH LEISHMANIASIS VAN DER AUWERA, G.1; BUFFET, P.2; BAILEY, M.3; BART, A.4; FELGER, I.5; BLUM, J.5
1.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 2.UMR945 IMMUNITY & INFECTION INSERM UPMC AND DEPARTMENT OF PARASITOLOGY, PITIÉ-SALPÊTRIÈRE HOSPITAL, PARIS, FRANCE; 3.BIRMINGHAM HEARTLANDS HOSPITAL & ROYAL CENTRE FOR DEFENCE MEDICINE, BIRMINGHAM, UNITED KINGDOM; 4.CENTRE FOR INFECTION AND IMMUNITY AMSTERDAM, ACADEMIC MEDICAL CENTRE, AMSTERDAM, NETHERLANDS; 5.SWISS TROPICAL AND PUBLIC HEALTH INSTITUTE, BASEL, SWITZERLAND.
Keyword:europe; travellers; patient management
Abstract: Except in the Southern Mediterranean area, human infection with Leishmania is uncommon in Europe. Typically infections occur during tourism or military activities, or when European citizens visit relatives and friends in endemic regions. As a result, relatively few cases are seen even in tertiary reference centres, the incidence being too small in each country to perform appropriately powered trials of antileishmanial treatment regimens. Moreover, patients from non-endemic regions may react differently to a certain infection or treatment as compared to patients who are frequently exposed to the parasite. Therefore compiling robust diagnostic and therapeutic data directly from patients attending European travel and military clinics is the only means to generate sufficient medically relevant information to support recommendations for the management of European patients with leishmaniasis. In addition, when travellers have visited several endemic areas, accurate determination of the infecting Leishmania species requires a globally applicable species typing approach to allow a more accurate prognosis and guiding therapeutic decisions.
A European consortium was established to build a joint database of clinical and diagnostic data and to collect biological reference material. This network is called LeishMan, short for Leishmaniasis Management, and currently consists of 31 members from 21 institutes in Belgium, France, Germany, The Netherlands, Portugal, Spain, Switzerland, and the UK. LeishMan aims at standardising procedures for diagnosis and parasite typing, and harmonising treatment guidelines. The LeishMan database permits systematic observation of cases from 2012 onwards. The research priority is on imported cutaneous leishmaniasis, but the database will cover all clinical leishmaniasis cases seen at the partner institutes. The consortium is currently seeking active participation of additional European countries, both where Leishmania is endemic and others. In the future, extension to non-European countries will be considered. Further information can be found on www.leishman.eu.
- GENETICS, EVOLUTION AND TAXONOMY [326] P 062 - PHYLOGENY OF LEISHMANIA SPECIES BASED ON THE HEAT-SHOCK PROTEIN 20 AND 70 GENES FRAGA, J.1; MONTALVO, A.M.1; VAN DER AUWERA, G.2; MAES, I.2; DUJARDIN, J.2; REQUENA, J.M.3
1.DEPARTAMENTO DE PARASITOLOGÍA, INSTITUTO DE MEDICINA TROPICAL PEDRO KOURÍ, HAVANA, CUBA; 2.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 3.CENTRO DE BIOLOGÍA MOLECULAR "SEVERO OCHOA" (CSIC-UAM), MADRID, SPAIN.
Keyword:heat-shock protein; phylogeny; typing
Abstract: The Leishmania genus comprises up to 35 species, of which 20 are responsible for human disease, some with a taxonomic status still under discussion. Heat-shock proteins (HSPs) play an important role in folding, assembly, intracellular localization, secretion, regulation, stabilization and degradation of other proteins. These proteins are amongst the most highly conserved proteins across the evolutionary tree. In Leishmania, the genes encoding HSP70 are widely applied for species identification based on restriction-fragment length polymorphisms, and typing or phylogenetic studies based on sequence comparisons. However, the use of additional molecular markers would be desirable to aid in establishing the phylogenetic relationships within the genus Leishmania. The small HSPs have been extensively studied because of their importance in protecting cellular proteins from aggregation, and maintaining cellular viability under intensive stress conditions, which is particularly important for microorganisms suffering dramatic temperature changes. In spite of their functional conservation, this class of HSPs has lower sequence conservation than HSP70. In Leishmania, only one protein of this family has been described, the 20 kDa heat-shock protein (HSP20). In the present study, we used hsp20 and hsp70 genes as targets to investigate the phylogenetic relationships among Leishmania species. The study combined new and publically available sequences corresponding to a 370-bp coding fragment of hsp20 and a 1380-bp coding fragment of the cytoplasmic hsp70. A total of 41 strains of different geographic origin, and including 16 different Leishmania species were analyzed. Phylogenetic trees were constructed using neighbor joining, maximum parsimony and maximum likelihood methods. The ratio of non-synonymous (dN) to synonymous (dS) substitutions in hsp20 and hsp70 were dN/dS < 1 by comparing Leishmania species, suggesting that these genes have been subject to purifying selection for the conservation of functions within the genus. Analysis of the combined dataset of both these genes confirmed the existence of the two distinct subgenera L. (Leishmania) and L. (Viannia) in the genus Leishmania. The phylogenic tree allowed the separation of the Leishmania genus into nine species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis. Additionally the (sub
)species L. (L.) infantum and L. (V.) peruviana were recognized. This study adds to the existing data for establishing a better and more consequent taxonomy of the genus Leishmania. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [333] P 063 - MODULATORY PROPERTIES OF DENDRITIC CELL PRIMED KINETOPLAST MEMBRANE PROTEIN ANTIGEN (KMP-11) FOR AN UNREGULATED PROTECTIVE TH1 RESPONSE IN INDIAN KALA-AZAR PATIENTS CHAUDHARY, R.; AMIT, A.; YADAV, A.; KUMAR, V.; DAS, P.; BIMAL, S. RMRIMS-ICMR, PATNA, INDIA.
Keyword:sla: soluble leishmania antigen; apcs- antigen presenting cells; tgf-b: transforming growth factor-beta,
Abstract: Kinetoplastid membrane protein antigen was recovered from its gene cloned in pQE-30 plasmid by extraction of Histidine tagged protein through Ni-NTA affinity chromatography. The bound protein was eluted in presence of 1mM imidazole concentration. This recombinant antigen was used to prime 1x106 monocyte derived dendritic cells (CD1a+ cells) and macrophages (CD14+ cells) from patients and healthy control individuals and changes in cytokines (IL-12, IL-10 and TGF-B) released from APCs were examined along with investigating transcription of NF-κB and Smad-4. These results in the APCs were co-related with Cytokine polarisation index in Tcells of VL patients. The results demonstrated that in respect to moDCs, the recombinant KMP11 protein was able to trigger 1.84 fold more IL-12 alone and about 1.95 fold IL-12 when given along with SLA. Further investigations carried out to reveal changes in immuno-suppressive cytokines demonstrated that KMP-11 primed macrophages producing IL-10 and TGF-B was significantly higher in patients than KMP-11 primed dendritic cells. The differential production of IL-12 between macrophages and dendritic cells was observed mainly due to differences in NF-κB production after the presentation of recombinant KMP-11 antigen. The cytokine bias (IFN-γ Vs IL-10) after stimulation of dendritic cells with recombinant protein was more towards IFN-γ with less induction of IL-10. Such alterations in cytokine production in respect to KMP11 antigen which we have observed in this study can contribute to bias the immune response which may be important for the outcome of the disease. - CELLULAR AND MOLECULAR BIOLOGY [335] P 064 - THE IMPACT OF LAURIC ACID IN THE CULTIVATION OF LEISHMANIA INFANTUM: ITS EFFECT ON GROWTH OF THE PARASITES BRUNO, F.; VITALE, F.; CASTELLI, G.; SCOPELLITI, D.; LUPO, T.; MARGIOTTA, A.; PIAZZA, M. CENTRO REFERENZA NAZIONALE LEISHMANIOSI - ISTITUTO ZOOPROFILATTICO SPERIMENTALE DELLA SICILIA, PALERMO, ITALY.
Keyword:lauric acid; cell growth; leishmania
Abstract: Lauric acid is a saturated fatty acid with a 12-carbon atom chain, thus falling into the medium chain fatty acids, is a white, powdery solid with a faint odor of bay oil or soap. Lauric acid, as a component of triglycerides, comprises about half of the fatty acid content in coconut oil, laurel oil, and in palm kernel oil (not to be confused with palm oil) Otherwise it is relatively uncommon. It is also found in human breast milk (6.2% of total fat), cow's milk (2.9%), and goat's milk (3.1%). Lauric acid has an antimicrobial activity against some pathogens as Salmonella enteritidis, Escherichia coli, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus. The objective of this study was to investigate the in vitro activities of
Lauric acid against protozoa Leishmania infantum. Leishmania infantum promastigotes were plated into 25 cm2 flasks containing RPMI-PY medium supplementing with FCS (10%) and 1% Glutamine and treated with scalar concentration of compound. The authors proceeded to the evaluation of the percentage of viability of Leishmania, by cell counting and comparison with the control culture (100% viability) after 48 hours treatment at 24 ° C. Experimental data has been collected showing a dose-dependent growth of Leishmania infantum treated with Lauric acid. In order to evaluate the action of lauric acid was also evaluated in cultures of Leishmania panamensis, and was performed a MTT viability assay on immortalized cell lines DH82. After 48 hours the results showed no toxic action of the compound in Leishmania and cell lines DH82, suggesting its possible use in culture media and to facilitate the isolation of the parasite. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [336] P 065 - PROTEOMIC PROFILE OF A LUTZOMYIA LONGIPALPIS-DERIVED (LULO) CELL LINE: EVIDENCES FOR A PROMISING NOVEL IN VITRO MODEL FOR LEISHMANIA-HOST INTERACTION STUDIES CÔRTES, L.M.C.1; CORRÊA, P.R.1; SOARES, R.O.A.1; PEREIRA, B.A.S.1; WAGHABI, M.C.1; LIMA, L.M.1; BELLO, F.J.G.2; BRAZIL, R.P.1; ALVES, C.R.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSIDAD DEL ROSARIO, BOGOTÁ, COLOMBIA.
Keyword:lulo cells; leishmania-host interaction ; proteomic
Abstract: The leishmaniases are diseases caused by protozoan of the genus Leishmania, transmitted by phlebotomine vectors and are currently considered diseases with increasing incidence in Brazil. Little information is available about the Leishmania adhesion molecules responsible for interaction with cells obtained from the phlebotomine sand flies. Most of the in vitro studies regarding Leishmania spp adhesion, invasion and differentiation have been performed with macrophages or fibroblast cultures. Our present results show that a cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae), named Lulo, could be an alternative model for studying leishmania adhesion to its invertebrate host. Our objective is to describe the proteins from the Lulo whole cell extract, based on a combined approach of two-dimensional gel electrophoresis (2DE) analysis and mass spectrometry (MS) protein identification. Lulo cells were maintained in Grace/ L-15medium and after 48 hours of culture, cells were harvested for protein extraction with RIPA lysis buffer and, then, retrieved for proteomic studies. Approximately 500 g of the protein extracts were subjected to 2DE and stained with Coomassie Colloidal. About 320 spots were detected in the 2DE images, and from these, 84 could be identified by MS using a MALDI TOF-TOF 4700 Proteomics Analyser. Identified spots included cyclophilin, enolase, putative Cu/Zn superoxide dismutases, between others. This study presents the first proteomic analysis of Lulo cells and represents a step towards the establishment of a new model in order to study of the interaction between Leishmania and its vector insect. - GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [348] P 066 - IDENTIFICATION OF LEISHMANIA INFANTUM VIRULENCE FACTORS BY PROTEOMIC APPROACH PIRES, S.F.; FIALHO JÚNIOR, L.C.; DE SOUZA, C.C.; TAFURI, W.L.; MELO, M.N.; ANDRADE, H.M. UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:l. infantum chagasi; virulence; proteoma
Abstract:
Leishmania infantum chagasi is the visceral leishmaniasis (VL) agent in Brazil, a zoonotic disease with high morbidity and mortality with the dogs being the main reservoir. VL canine is a severe disease which the symptoms are present in less than 50% of infected dogs. Clinical variability suggests that parasite factors are involved in virulence. We used two L. infantum chagasi strains: MHOM/BR/1972/BH46 (BH46) and MCAN/BR/2000/BH400 (BH400) with high and low virulence, respectively. We realized in vitro and in vivo virulence characterization these strains by infecting macrophages and hamsters, respectively. These assays had shown a statistically significant bigger parasitism in cells and animals infected with BH400 than BH46 strain. Furthermore, we used proteomic approach to identify proteins that may be involved in virulence difference. The protein extracts were obtained from promastigotes forms and fractionated by electrophoresis two dimensional DIGE (Differential Gel Electrophoresis ) using 18 cm pH 4-7 strips and 12% SDS-PAGE. The gel images were analyzed by DeCyder® software (GE Healthcare, USA). The analysis showed 13 spots with higher expression in BH400 than in BH46. No overexpressed spots were identified in BH46 when compared to BH400. The differential expressed spots were identified by Mass Spectrometry (MALDI/ToF-ToF) (Bruker Daltonics, Billerica, USA). Among them are α and β-tubulins, heat shock protein 83-1, paraflagellar rod protein 2C, putative cysteine peptidase, enolase, putative adenosine kinase, translation elongation factor 1-β, putative NADP-dependent alcohol dehydrogenase, putative proteasome activator protein pa26, kinetoplastid membrane protein-11, glutaredoxin-like protein and conserved hypothetical protein. Some the identified proteins are described as virulence factor in literature and their role in virulence phenotype will be evaluated by transgenic parasites. The virulence factor studies may contribute to the discovery of new targets with therapeutic potential against leishmaniasis and in parasite-host interaction. Financial support: FAPEMIG, CAPES and CNPq
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [349] P 067 - EVALUATION OF HISTOPATHOLOGICAL PARAMETERS AND CYTOKINES PROFILE IN SPLEEN OF HAMSTERS (MESOCRICETUS AURATUS) EXPERIMENTALLY INFECTED WITH TWO STRAINS OF LEISHMANIA (LEISHMANIA) INFANTUM WITH DIFFERENT DEGREES OF VIRULENCE AND PATHOGENICITY MOREIRA, N.D.1; SOUZA, J.V.1; ROATT, B.M.1; VIEIRA, P.M.A.1; REZENDE, M.T.1; MATHIAS, F.A.S.1; CARDOSO, J.M.O.1; LANA, M.1; OLIVEIRA, R.C.2; SÁ, R.G.1; GIUNCHETTI, R.C.1; CARNEIRO, C.M.1; BOUILLET, L.M.1; REIS, A.B.1
1.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL.
Keyword:hamster; visceral leishmaniasis; cytokines
Abstract: The hamster model (Mesocricetus auratus) is considerate a good experimental model to study visceral leishmaniasis (VL), since the infection in this experimental model reproduces several typical aspects of the human visceral leishmaniasis (HVL) and canine leishmaniasis which are closely related with the inoculation route. However, little is known in relation to the evolution of the natural history during the experimental infection by L. infantum in hamsters as well as the use of this model to evaluate vaccine and/ or drugs in pre-clinical therapeutic study. In the present work, we determined through the quantitative Real Time PCR technique the mRNA expression of cytokines (IL-10, TGF-β, IFN- and TNF-α) in the spleen of hamsters experimentally infected by different routes (intradermal, ID; intraperitoneal, IP; and intracardiac, IC) and strains of L. infantum (MHOM/BR/74/PP75 and Wild). Moreover, the histopathological changes were evaluated and the animals were also followed at 1, 3, 6 and 9
months post-infection. Our major results showed that the histopathological changes in the spleen of animals infected with PP75 strain were more evident in the IC group with greater intensity in the final periods of infection (6 and 9 months). However, in animals infected with the Wild strain, changes in IC group occurred throughout the study period, but with more exuberance from the sixth month. Interestingly, it could be observed a predominant expression for IFN- in hamsters infected with PP75 strain at the end of evaluation (6 and 9 months) in IC group, suggesting higher expression of this cytokine in response to the parasite replication inducing an intense inflammatory profile. On the other hand, the cytokine immune response observed in hamsters infected with the wild strain in the IC group during the initial period (3 months) was related to highly increased expression of IL-10 and TGF-β. These findings appear to suggest suppression of these animals and the establishment of the active infection and parasitic tissue proliferation. Therefore, according the results obtained in the present study, it is possible to suggest the use of hamsters as experimental model to evaluate the immunopathological findings during ongoing active VL. Considering that the infected hamster presented many of the immunopathological features as observed in HVL and canine visceral leishmaniasis (CVL), we recommend this model for preclinical evaluation of antileishmanial drugs and vaccine.] "Supported by: CAPES, FAPEMIG, CNPq e UFOP" - VACCINE DEVELOPMENT AND TRIALS [352] P 068 - LIESP/QA-21 (CANILEISH®) VACCINE INDUCES POSITIVE DTH RESPONSES AFTER THE PRIMARY VACCINATION AND ONE YEAR LATER PAPIEROK, G.M.1; BUTAUD, T.1; VOULDOUKIS, I.2; MARTIN, V.1; CUISINIER, A.1; GUEGUEN, S.1 1.VIRBAC, CARROS, FRANCE; 2.INSERM UPMC-UMRS945, PARIS, FRANCE.
Keyword:dth; skin test; vaccine
Abstract: The Canine Leishmaniasis vaccine CaniLeish® composed of purified L. infantum Excreted Secreted Proteins (ESP) was developed in the late 20th and early 21st Centuries. Several studies (1-4) constitute the basis of this innovative vaccine. This study was performed in order to check the duration of immunity of CaniLeish®. 18 dogs received 3 subcutaneous injections of one vaccine dose at 3-week intervals (D0, D21 and D42) and 18 other dogs were kept as controls. A leishmanin skin test was performed 3 weeks after the last vaccine injection (D63) on 8 vaccinated dogs and 8 controls, and also one year later (week 58) on the rest of the dogs. The delayed-type hypersensitivity (DTH) test local reactions were followed daily for one week. Canine Macrophage Leishmanicidal Assay (CMLA) and serological follow up was also performed. The serological analyses performed before vaccination confirmed that all the dogs were free from antibodies against the vaccinal proteins (ESP and Parasite Surface Antigen PSA) before the 1st injection, and all the control dogs remained free from these antibodies throughout the study. The skin tests performed three weeks and one year after vaccination demonstrated the presence of a memory cell-mediated immune response by the observation of a positive DTH response. This was associated with a predominantly IgG2 antibody response lasting less than 6 months after the primary course. There was a small but rapid memory response after the DTH test performed on week 58.
All vaccinated dogs had a positive CMLA result by the time of the DTH test, whereas no control dog reached the threshold result in this test at any point. (1) J.L. Lemesre et al “Protection against experimental visceral leishmaniasis infection in dogs immunized with purified excreted secreted antigens of Leishmania infantum promastigotes”. Vaccine 23 (2005) 2825-2840. (2) J.L.Lemesre et al “Long-lasting protection against canine visceral leishmaniasis using the Li ESAp-MDP vaccine in endemic areas of France : double-blind randomised efficacy field trial ». Vaccine 25 (2007) 4223-4234. (3) J. Moreno et al. Use of a LiESP/QA-21 Vaccine (CaniLeish) Stimulates an Appropriate Th1Dominated Cell-Mediated Immune Response in Dogs. PLoS Negl Trop Dis 6(6): e1683 (4) Bongiorno et al. Vaccination with LiESP/QA-21 (CaniLeish®) reduces the intensity of infection in Phlebotomus perniciosus fed on Leishmania infantum infected dogs – a preliminary xenodiagnosis study (submitted for publication)
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [360] P 069 - ASSESSMENT OF PHENOTYPIC AND FUNCTIONAL MONOCYTES PROFILE IN PATIENTS WITH CUTANEOUS LEISHMANIASIS BEFORE, 30 AND 90 DAYS AFTER TREATMENT WITH N-METHYL GLUCAMINE ALVES, M.L.R.1; COSTA E SILVA, M.F.2; PASCOAL-XAVIER, M.A.3; ZAULI, D.A.G.2; DOSREIS-JUNIOR, C.A.2; CUNHA, G.M.R.2; MARTINS-FILHO, O.A.2; TEIXEIRA-CARVALHO, A.2; RIBEIRO-DE-SENNA, M.C.2; FARIA, A.C.2; RABELLO, A.2; PERUHYPE-MAGALHÃES, V.4 1.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, AVENIDA AUGUSTO DE LIMA 1715, CEP:30190002, BELO HORIZONTE, MG, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 3.FACULDADE DE MEDICINA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 4.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:cutaneous leishmaniasis; phagocytosis by human monocytes; immunological profile
Abstract: Localized Cutaneous Leishmaniasis (LCL) is the most common clinical manifestation caused by Leishmania (V.) braziliensis infection. There are few studies assessing the immunological events associated with the interaction Leishmania/phagocytes in human CL. In this study, we evaluated the phenotypic and functional peripheral blood monocytes profile of 10 healthy volunteers and 8 patients with 1-3 cutaneous lesions, with 45 days to 4 months of disease. All patients were referred to the Leishmaniasis Reference Center - CPqRR and evaluated at first assessment (CL T0), 30 (CL T30) and 90 days (CL T90) after treatment. By employing a system of immunophenotyping multicromatic platform, it was possible to establish a protocol for multiparametric analysis of phagocytosis and to assess simultaneously the expression of surface markers (IL-10r, IFN-r, TLR-2, TLR-4, HLA-DR, CD32, CD64, CD80 and CD86) and the profile of intracellular cytokines production (TNF-α, IL-12, IL-10, TGF-β) and NO by monocytes. The samples were cultured in the presence or absence of L. (V.) braziliensis and in the presence or absence of autologous plasma. The data indicate that before treatment (CL T0), circulating monocytes showed good phagocytic capacity associated with high expression of TLR-2 and TLR-4, CD16, CD80 and IFN-r and increased production of TNF-α, IL-12 and NO. Concomitant to this inflammatory profile, regulated expression of CD32, CD86 and HLA-DR, and the high expression of IL-10r were observed, in combination with the increased production
of IL-10 and TGF-β, important modulating cytokines. After 30 days of treatment (CL T30), there was a tendency to the modulation of inflammatory immune response, with increased expression of IL-10r and reduction in the expression of TLR-2, CD16, CD80 and HLA-DR associated with a regular/normal or reduced TNF-α, IL-12, IL-10, TGF-β and NO production. The immunological profile observed after 90 days of treatment is similar to the observed for the healthy individuals. Regular/normal expression was observed in most receptors of monocytes surface associated with regular/normal production of cytokines and NO. Corresponding Author:
[email protected] Financial Support: FAPEMIG, CNPq
- CELLULAR AND MOLECULAR BIOLOGY [377] P 070 - POST-TRANSCRIPTIONAL REGULATION OF LEISHMANIA AQUAGLYCEROPORIN AQP1 MANDAL, G.1; MANDAL, S.1; ORTA, J.1; PAPADOPOULOU, B.2; MUKHOPADHYAY, R.1 1.FLORIDA INTERNATIONAL UNIVERSITY, MIAMI, UNITED STATES; 2.UNIVERSITY OF LAVAL, QUEBEC, CANADA.
Keyword:aquaglyceroporin; regulation; 3'utr
Abstract: Leishmania aquaglyceroporin 1 (AQP1) is responsible for important physiological functions such as volume regulation and osmotaxis as well as drug (trivalent antimony, SbIII) sensitivity. However, the mechanism(s) of regulation is largely unknown. In the absence of definitive promoter and transcriptional control Leishmania depends on post-transcriptional and/or posttranslational control for gene regulation. Recently we reported that at post-translational level Leishmania mitogen activated protein kinase2 (MPK2) regulates AQP1 stability through phosphorylation. However, post-transcriptional regulation of this important drug transporter is completely unknown. AQP1 mRNA contains a long 3’UTR (~ 1.8 kb). The 3’UTRs are established components of post-transcriptional regulation. They influence the mRNA stability as well as translational efficiency. The AQP1 U-rich 3’UTR contains several ARE (AU rich sequences) and CURE (CU rich sequences) motifs that regulate mRNA stability in higher eukaryotes. The stability studies revealed that AQP1 mRNA is unstable. The role of different controlling elements in the 3’ UTR for AQP1 regulation will be discussed. - OTHER SPECIAL TOPICS [381] P 071 - PROFILE OF INTRACYTOPLASMIC CYTOKINE SYNTHESIS IFN-Γ AND IL-4 BY T LYMPHOCYTES (CD4+ AND CD8+) IN PERIPHERAL BLOOD OF DOGS NATURALLY INFECTED BY LEISHMANIA INFANTUM LEAL, G.G.A.1; COURA-VITAL, W.2; ROATT, B.M.1; AGUIAR-SOARES, R.D.1; GIUNCHETTI, R.C.2; CARNEIRO, C.M.1; TEIXEIRA-CARVALHO, A.3; CARNEIRO, M.2; REIS, A.B.1
1.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.CENTRO DE PESQUISAS RENÉ RACHOU, BELO HORIZONTE, MG, BRAZIL.
Keyword:biomarkers; leishmania infantum; cytokine
Abstract: The search for immunological biomarkers is essential for understanding the mechanisms of resistance and susceptibility to canine visceral leishmaniasis. To evaluate the immunophenotyping and intracytoplasmatic cytokine synthesis IFN-γ and IL-4 by T cells
(CD4+ and CD8+) before and after stimulation with antigen of soluble L. infantum (SLA) in vitro, a total of 124 dogs naturally infected by L. infantum were assessed. The dogs were clinically classified, according to presence/absence of infection signs and serological and molecular tests: Asymptomatic Dogs I (AD-I, n= 34), with no suggestive signs of disease, negative serology and positive PCR for Leishmania; Asymptomatic Dogs II (AD-II, n= 20) positive serology; symptomatic (SD, n= 42), with characteristic clinical signs of CVL and positive serology; and Control Dogs (CD, n= 28), classified according to negative serological and PCR for Leishmania and absence of clinical signs. The results of intracytoplasmic synthesis of IFN-γ in the control culture AD-II animal group showed an increased production of T lymphocytes CD4+ IFN-γ+ compared to CD, AD-I and SD groups, and SD group had an increase compared to the CD group. In the stimulated culture, an increase in AD-II and SD groups was obtained compared to CD and AD-I groups. It was observed in evaluating the T lymphocytes CD8+ IFN-γ+ in control and stimulated culture that AD-II and SD animals groups showed an increase in the synthesis of IFN-γ when compared to the CD and AD-I groups. When evaluating the profile of IL-4 intracytoplasmic, there was an increase of T lymphocytes CD4+ IL-4+ in the control culture in AD-II group in relation to CD and AD-I groups. In the stimulated culture an increase in AD-II and SD groups compared to CD group was observed. When the production of T lymphocytes CD8+ IL-4+ was evaluated, it was observed in this case an increase of this cytokine in the AD-II and SD groups in relation to CD and AD-I groups in the control and stimulated culture. According to the disease progress our results suggest a greater synthesis of IFN-γ and IL-4 by subpopulations of T lymphocytes (CD4+ and CD8+) in the control culture and stimulated culture with SLA, giving a profile of immune mixed response for the animals. The AD-I group animals did not differ from CD animals group in the intracellular synthesis of these cytokines. Supported by: FAPEMIG, CNPq, PPSUS/MS and DECIT/MS - VACCINE DEVELOPMENT AND TRIALS [391] P 072 - RECRUITMENT AND CELL MIGRATION IN BLOOD MICE SENSITIZED WITH THE ADJUVANTS: GLA-SE, COMPLETE FREUND'S ADJUVANT, RESIQUIMOD, MONTANIDE PET GEL A AND AL(OH)3 MATHIAS, F.A.1; SOUZA, J.V.; MOREIRA, N.D.1; VIEIRA, P.M.A.1; CARDOSO, J.M.O.1; ROATT, B.M.1; AGUIAR SOARES, R.D.O.1; REZENDE, M.T.1; GIUNCHETTI, R.C.2; CARNEIRO, C.M.1; MELO, M.N.2; REIS, A.B.1 1.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:adjuvants; cell migration; mice
Abstract: An effective early innate immune response to vaccine adjuvants may significantly impact the overall immunogenicity and efficacy of vaccines. In this context, adjuvants are important additives, since they enhance the immunogenicity of an antigen by aiding the formation of an intense and prolonged immune response. Herein, inflammatory reaction and inflammatory cells influx in the peripheral blood induced by adjuvants were analyzed at 1, 12, 24, 48, 96, 168 and 336 hours after sensitization. Each animal received a single dose of adjuvants on the back and the selected adjuvants were Complete Freund’s Adjuvant (ACF), Glucopyranosyl Lipid Adjuvant–stable emulsion (GLA-SE), Montanide Pet Gel A ® (MPGA), Al(OH)3 and Resiquimod. Animals were inoculated with saline as a control group. The results of the white blood cell quantification showed that adjuvants were able to induce increases of total leukocytes population in peripheral blood in late periods as well as in differential analysis. In relation to systemic response through the immunophenotyping of circulating leukocytes from peripheral blood, the data pointed reduction in circulating T CD4+ lymphocytes in the ACF at 12h, in the GLA-SE group at 24h and Resiquimod group at 48h compared to control group. The MPGA group showed a slower reduction at 96h in relation to control. Concerning T CD8+ lymphocytes
population only the MPGA group showed a reduction at 24h as the Resiquimod group, but this one with an increase at 96h. In the analyses of B CD19+ lymphocytes an initial decrease in all groups during the early periods was observed. The GLA-SE and Al(OH)3 groups presented an increase at 24h and the ACF, MPGA and Resiquimod increased at 48h. The NK cell CD49b+ analyses showed a remarkable decrease in the Resiquimod group at 12h. The CD14+ monocytes analyses pointed an increase in the Al(OH)3 at 24 h and in the GLA-SE and MPGA at 48h and 96h respectively. Interestingly, the Resiquimod group showed an increase at 12h followed by a decrease in late periods. The cellular recruitment study promoted by the adjuvants is critical, since the activation of the innate immune response mediated by different cell types is extremely important to direct the adaptive immune response. Our data showed that the inflammatory response observed in the various vaccine adjuvants is mainly due to the nature of the adjuvant and the way it acts in immune system activation. Supported by: CNPq, FAPEMIG, CAPES and UFOP. - GENETICS, EVOLUTION AND TAXONOMY [399] P 073 - MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF L. (VIANNIA) STRAINS WITH ISOENZYMATIC HYBRID PROFILES BOITE, M.C.; SANTOS, B.N.; FERREIRA, G.E.M.; TRANNIN, M.A.; OLIVEIRA, T.S.; ALMEIDA, R.P.; CUPOLILLO, E. FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:hybrids; leishmania (viannia); recombination
Abstract: Leishmania hybrids were described mainly by Multilocus Enzyme Electrophoresis (MLEE); different phenotypes, compared to possible parental, were also pointed. In Multilocus Sequence Analysis (MLSA) incongruence between loci is detected. These sights suggest borderline strains (in terms of species delimitation) do occur. However, it is difficult to link MLEE hybrid profile with other mosaic characteristic. Here we aim to characterize by growth curve, in vivo infection and MLSA, three strains with hybrid profile detected by 6PGDH, an enzymatic system in MLEE: L. naiffi/L. braziliensis (Ln/Lb), L. guyanensis /L. braziliensis (Lg/Lb), and L. lainsoni/L. naiffi (Ll/Ln). For the enzymes G6PDH, ME, IDH nadp and GPI, the profile corresponded to one putative parental: L. naiffi, L. guyanensis and L. lainsoni. Golden hamsters were infected with 1x105 cells of hybrids and possible parental; all presented lesions after 15 days. The hamsters’ isolates kept the same isoenzymatic profile. The hybrids growth curves were similar to those observed for one of the putative parental, exactly as in MLEE. BLAST using nucleotides sequences of 6PGD, G6PD, MPI, ICD, HSP70, nuclear MDH and mitochondrial MDH, detected that (i) Ln/Lb hit with the same identity and Max score with L. braziliensis, L. lainsoni and L. naiffi for 6PGD; (ii) Lg/Lb presented higher identity with L. shawi in ICD (100% against 99% L. guyanensis); (iii) Ll/Ln presented higher identity and/or Max score with L. lainsoni for all markers. These results corroborate our previous observation: some alleles are shared across species in 6PGD, and L. shawi belongs to the L. guyanensis group. Recombination analysis in RDP with 120 L. (Viannia) strains, detected an interval of recombination in MDHmt locus for Ll/Ln, with minor and major parental being Ln/Lb and one L. braziliensis; and in G6PD for Lg/Lb, with minor and major parental being L. lainsoni and L. guyanensis. In order to clarify the occurrence of recombination process, its propagation and contribution to parasite evolution it is important to understand why some markers detect such variability and others do not, and why the hybrid aspect has not an equal inheritance patter of each parental. For such purpose, phenotypic and molecular approaches must still be performed with these strains. These previous results reflect that these so called hybrids by isoenzymes do represent uncommon strains, e.g. genetic mosaics or non-symmetric hybrids.
- CELLULAR AND MOLECULAR BIOLOGY [400] P 074 - OVEREXPRESSION OF TRANSLOCATOR PROTEIN (TSPO) IN J774 CELLS REDUCES PHAGOCYTOSIS OF LEISHMANIA AMAZONENSIS SIMÕES DIAS, B.R.1; GUEDES, C.E.S.1; CRUZ, K.P.1; ALMEIDA, N.J.1; VERAS, P.S.T.2 1.CENTRO DE PESQUISA GONÇALO MONIZ, FIOCRUZ, SALVADOR, BA, BRAZIL; 2.CENTRO DE PESQUISA GONÇALO MONIZ, FIOCRUZ-BA; INCT-DT, SALVADOR, BA, BRAZIL.
Keyword:tspo; leishmania; macrophage
Abstract: Leishmaniasis is a major neglected disease and a serious public health problem. Mouse models are used to understand mechanisms that underlie outcome of Leishmania infection. Macrophages are the main host cell for Leishmania infection. Previously, we demonstrated that macrophages from CBA mice control infection by L. major yet are permissive to L. amazonensis. In order to identify potential targets involved in Leishmania infection, we used a proteomic approach. Translocator Protein (TSPO) was found to be down-regulated during L. amazonensis infection in comparison with L. major infection. We hypothesized that TSPO modulation leads to control of L. amazonensis infection. Thus, this study aims to evaluate the role TSPO plays in Leishmania amazonensis infection. Initially, total RNA from L. majorinfected macrophages was purified and used to obtain TSPO-encoding gene using RT-PCR. Then, cloning of TSPO amplicon into pcDNA6.2/C-EmGFP-DEST was performed employing GATEWAY™ system to generate the plasmid pcDNA6.2-TSPO. The TSPO cloning was confirmed by sequencing and alignment with the TSPO gene sequence available in GenBank. Subsequently, pcDNA6.2-TSPO and pcDNA6.2 were used to stable transfect the macrophage cell-line J774 obtaining, respectively, J774 cells overexpressing TSPO and control mocktransfected cells. Using western blot analysis, TSPO expression was found to be increased by 20% in TSPO transfected J774 cells in related to control cells. In J774, overexpression of TSPO reduced by 59,77% the percentage of L. amazonensis-infected cells compared to control cells. This reduction was only observed at 6 hours after infection. TSPO-overexpressing cells also reduced in 26,2% phagocytosis index of latex beads, suggesting TSPO plays a role in macrophage cells-line phagocytic capability. Further studies using ligands-induced TSPO modulation will be conducted to evaluate the actual role TSPO plays in L. amazonensis infection. Supported by FIOCRUZ and CNPq - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [404] P 075 - LST AS AN IN VIVO MIRROR OF IFN-Γ PRODUCTION IN CANINE LEISHMANIASIS RODRÍGUEZ-CORTÉS, A.1; MARTÍNEZ-FLÓREZ, A.1; NADAL, J.M.2; TONÍN, L.2; LLULL, J.2; ALBEROLA, J.1
1.UNIVERSITAT AUTÒNOMA DE BARCELONA, BELLATERRA (BARCELONA), SPAIN; 2.MON VETERINARI, MANACOR, SPAIN.
Keyword:leishmanin skin test; interferon-γ
Abstract: Despite the importance of canine visceral leishmaniasis, the immunological mechanism that determines illness or resistance still remains unclear. It is known that a balance between the different types of immune response is needed, and that the predominance of the Th1 cellmediated immunity (CMI) is associated to a protective response against the disease. The measure of the IFN-γ production can be used to meassure this Th1 CMI in vitro but it is an expensive and time consuming procedure. The leishmanin skin test (LST) is a delayed-type
hypersensitivity reaction to an intradermal injection of an inactivated suspension of promastigotes and constitutes an interesting candidate for easily evaluating the CMI in vivo. To the authors’ best knowledge, there are not studies that show a clear relationship between LST positive response and immuno-protection in naturally infected dogs. For this purpose, 69 dogs living in a highly endemic area (Mallorca) were included in the study. Clinico-pathological score (CPS), anti-Leishmania specific antibodies, LST reaction, and Leishmania-specific IFN-γ and IL10 levels were evaluated. Statistical analysis showed a significant correlation between LST diameter and IFN-γ levels (ρ = 0.408, P = 0.005). In addition, a significant greater LST and IFN-g response was found when comparing clinically healthy dogs (CPS < 7) with those clinically sick (P < 0.001). Leishmania-specific IL10 levels were not correlated with none of the other parameters. This is the first study showing a significant relationship between LST and IFN- γ, widely recognized as the most representative Th1 cytokine. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [405] P 076 - ANATOMOPATHOLOGICAL CHANGES ASSOCIATED WITH DEATH IN HUMAN VISCERAL LEISHMNIASIS AGUIAR, S.B.1; ALMEIDA, M.A.C.2; FREITAS, L.A.R.1; DOS-SANTOS, W.L.C.1 1.AFUNDAÇÃO OSWALDO CRUZ, CENTRO DE PESQUISA GONÇALO MONIZ, SALVADOR, BA, BRAZIL; 2.UNIVERSIDADE FEDERAL DA BAHIA, FACULDADE DE MEDICINA DA BAHIA, SALVADOR, BA, BRAZIL.
Keyword:lymphoid tissue; co-infection; autopsy
Abstract: Introduction: Visceral leihmaniasis is endemic in Brazil with an estimated lethality of 5-12%. Little is known on the mechanism leading to death in visceral leishmaniasis. However, these mechanisms may be expressed in tissue changes. Objective: The aim of this study is to investigate the tissue changes, including changes in cell populations and in the pattern of cytokine expression in patients that died of visceral leishmaniasis. Methods: A total of 21 necropsies of patients that died of visceral leishmaniais from 1975 to 2000 was identified in the autopsy archives of Professor Edgard Santos Hospital (Federal University of Bahia, Brazil). The necropsy reports were reviewed and demographic, clinical, laboratorial and macroscopic information collected. The necropsy slides were reviewed by two pathologists. Results: Among the patients 13 (62%) were male, age varied between 6 moths to 56 years with median of 10 years. The median time of disease was 6 months. Anemia was reported in 17 (81%) patients, edema in 13 (62%), ascites in 4 (19%), hemorrhage in 12 (57%), diarrhea in 11 (52%) and coinfections in 17 (81%). Albumin was low in 17/17 patients and the median of globulin/albumin ratio was 2. Serum creatinin was high in 7/16 patients, ALT was high in 10/15, AST in 10/15 and FA in 7/15. White blood cells count was low in 13/20 patients. Neutrophils were low in 8/12 patients. Hepatomegaly was present in 17/18, cholestasis in 7/18, splenomegaly was present in 19/20, pulmonary hemorrhage 4/21. The histological analysis of the slides of 8 autopsies made so far, revealed inflammatory infiltrates in liver (6/6), lungs (5/6) and kidneys (3/6). Lymphoid atrophy and disorganization of splenic lymphoid tissue was observed in 6/6 patients. Conclusion: Visceral leishmaniasis is associated with a high frequency of co-infection and splenic lymphoid tissue atrophy and disorganization. We are now completing the study of the autopsies and starting the studies on cell populations and cytokines by immunohistochemistry. PPSUS. Saulo Brandão [
[email protected]]
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [406] P 077 - CANINE VISCERAL LEISHMANIASIS AND SPLENIC PLASMA CELLS REDISTRIBUTION SILVA, J.S.1; SANTOS, T.C.1; DUNN-WALTERS, D.2; OLIVEIRA, G.G.S.1; DOS-SANTOS, W.L.C.1
1.FUNDAÇÃO OSWALDO CRUZ, CENTRO DE PESQUISAS GONÇALO MONIZ, SALVADOR, BA, BRAZIL; 2.KING’S COLLEGE LONDON, UNIVERSITY OF LONDON, LONDON, UNITED KINGDOM.
Keyword:spleen; lymphoid tissue; plasma cell
Abstract: Introduction: Visceral leishmaniasis is associated with disruption of splenic lymphoid tissue and redistribution of cell populations involved with the immune defense. In this work, we studied the alterations in distribution of different isotype of plasma cells in the spleen of dogs naturally infected with Leishmania infantum. We also made a database of canine germline genes repertoire in order to analyze in the future the clonal relation among different isotypes of plasma cells using Hight throughput Sequencing of different regions of CR3 of genes of immunoglobulin. Material and Methods: Spleen samples from dogs grouped into three categories: noninfected animals with organized white pulp, infected animals with organized white pulp and infected animals with disorganized white pulp, were used in the study. The sections were labeled with anti-dog IgG, IgM and IgA antibodies for plasma cells identification. The number and distribution of plasma cells were estimated. Additionally, the clinical and laboratory data (biochemistry and hematology) of the animals were reviewed. Available DNA linear sequence (GenBank accession number NW_003726071.1) was used to search for corresponding genomic sequences by looking for TATTACTGT or CACAGTG (part of RSS).Results: Plasmacytosis was greater in infected animals with disorganized white pulp (7/10) than in infected animals with organized white pulp (2/10, Chi-square, P < 0,04). The albumin/globulin ratio and the clinical score related to canine visceral leishmaniasis were higher in the animals with disorganized white pulp in comparison with the animals with organized white pulp. A region of approximately 1.28Mb from dog chromosome 8 was found to contain potential VH, DH and JH gene segments. Ninety two VH, six DH and three JH segments were mapped to a 1.28Mb region of the dog chromosome 8. Conclusions: The plasmacytosis and disglobulinemia associated with visceral leishmaniasis may result of a disruption of the white pulp of the spleen, affecting B cell differentiation. The obtained canine database of immunoglobulin genes will help us to identify by the result of the Hight throughput Sequencing the clonal relation among different isotypes of plasma cells in spleen of different groups of dogs. FIOCRUZ-PAPES V, CNPq, CAPES, FAPESB.
[email protected] - GENETICS, EVOLUTION AND TAXONOMY [413] P 078 - POLYMORPHISM IN THE HASPB REPEAT REGION OF EAST AFRICAN LEISHMANIA DONOVANI STRAINS ZACKAY, A. HUJI, JERUSALEM, ISRAEL.
Keyword:haspb; k26; leishmania donovani
Abstract: Background/Objectives: Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected
patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission. Methodology/Principal findings: Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient’s geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL coinfected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions. Conclusions/Significance: HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [416] P 079 - EXOPROTEOME DYNAMICS IN LEISHMANIA INFANTUM SANTAREM, N.1; RACINE, G.2; SILVESTRE, R.1; CORDEIRO-DA-SILVA, A.1; OUELLETTE, M.2 1.INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR (IBMC) DA UNIVERSIDADE DO PORTO, PORTO, PORTUGAL; 2.CENTRE DE RECHERCHE EN INFECTIOLOGIE DU CENTRE DE RECHERCHE DU CHUL, QUÉBEC, CANADA.
Keyword:exoproteome; vesicles; gp63
Abstract: The exoproteome of Leishmania infantum is composed of parasite derived proteins present in the extracellular environment. Although the exoproteome might have a significant role in the precocious steps of infection little is known concerning its composition. We developed an approach enabling the in vitro recovery of the exoproteome from logarithmic and stationary L. infantum promastigotes. The recovered exoproteomes were then further separated into two fractions, vesicles and vesicle depleted exoproteome, evaluating the protein profile of each fraction. Although the most abundant protein in all fractions was GP63, the protein composition of the separated fractions was distinct reflecting the origin of the fraction and the metabolic state of the parasites. The vesicle-derived exoproteome recovered from logarithmic parasites was significantly enriched in ribosomal proteins, indicating a potential role for these vesicles in protein turnover. Also, a stage specific enrichment of vesicles with properties related to apoptotic vesicles was observed in stationary phase parasites and evidence was obtained that the release of vesicles was increased in response to a death stimuli. This report on the exoproteome obtained from in vitro promastigote cultures provides new perspectives on Leishmania biology with the possibility of vesicles playing a major role in protein turnover and also in cell death.
- OTHER SPECIAL TOPICS [418] P 080 - THE ISOMORPHIC PHENOMENON OF KOEBNER IN LEISHMANIASIS: A CASE REPORT SILVA JÚNIOR, C.F.; DILL, D.C.S.; SOUZA, P.R.; GUEDES, R.P.O.; CASTILHO, W.S.; KJAER, V.K. UNIVERSIDADE FEDERAL DE RONDÔNIA, PORTO VELHO, RO, BRAZIL.
Keyword:leishmaniasis; koebner phenomenon; trauma
Abstract: Introduction: Kobner phenomenon consist in the occurrence of a pre-existing disease in normal skin subjected to a trauma and it is usually associated with non-infectious diseases such as: psoriasis, vitiligo and lichen planus, but it has been reported in patients with American Cutaneous Leishmaniasis. This fact suggests the existence of an asymptomatic systemic infection or a latent one, reflecting the existence of systemic or local parasitized macrophages circulating. Results and discussion: Patient, aged 30, male, came to a consult on December 2012 in the dermatology´s ambulatory of Specialized Center of Tropical Medicine of Rondônia (CEMETRON), reporting that on August 2012 suffered an injury in the right eye, due to a direct trauma with a stone. He looked for several medical cares with ophthalmologists, starting multiples treatments, but without satisfactory results. During physical examination he presented two scars with a parchment/atrophic aspect in the left buttock and complaints of erythema, decreased of the visual acuity, photosensitivity visual and photophobia in the right eye. Ophthalmologic examination revealed multiple elevated lesions in the anterior stroma, white, irregular and with indefinite borders, located at the apex of the cornea. He reported that 8 months ago presented two ulcers in the region of the left buttock that healed spontaneously within 3 months. In consultation on December 12th 2012, due to the suspicious lesions and because he lives in an endemic region, was requested Polymerase Chain Reaction (PCR) exam as of both healed lesions of left buttock as of the secretion of ocular region to search for leishmanias, and the later result of this exam was positive. The appearance of a corneal ulcer after mechanical injury in a patient with untreated American Cutaneous Leishmaniasis allowed the occurrence of the Koebner phenomenon. This patient had his treatment initiated on January 8th 2013, with N-methyl meglumine antimoniate (Glucantime™) in dose of 20mg SbV/kg/day, during 30 days. Conclusion: We emphasize the unusual and inedited aspect of this type of ophthalmologic injury complicating cases of American Cutaneous Leishmaniasis, being also important to warn the patients about the possibility of occurrence of this intercurrent and should be recommended care with any kind of trauma in other body regions.
- GENOMICS, TRANSCRIPTOMICS, PROTEOMICS, METABOLOMICS [423] P 081 - IDENTIFICATION OF PHOSPHOLIPIDS TRANSLOCATORS IN LEISHMANIA AMAZONENSIS JORGE, C.L.; MUXEL, S.M.; DOS SANTOS, M.G.; FLOETER-WINTER, L.M. INSTITUTO DE BIOCIÊNCIAS USP, SÃO PAULO, SP, BRAZIL.
Keyword:apoptosis; genomic library; annexin-v
Abstract: When inoculated by the insect in the host mammal, some promastigotes of Leishmania presents a strategy of apoptotic mimicry by changing the lipids composition of the external leaflet in the plasma membrane, as a result from the translocation of phospholipids to the external face of the lipid bilayer. The macrophages recognize this apoptotic signals and perform the phagocytosis of the parasites without launching an inflammatory process. Here we described a strategy that explores the binding of annexin V to the exposed phospholipids to identify the mechanism responsible for this exposure in Leishmania (L.) amazonensis. Wild-type promastigotes were transfected with a genomic library of this organism cloned in a shuttle cosmid containing the hygromicin resistance marker. This way, transfected mutants will carry extra copies of segments of the Leishmania genome. The transfectants carrying the information for the lipid exposure should be able to bind annexin-V in the external membrane in an earlier development stage than normal. The transfectants were labeled with annexin V-FITC and the cells with increased fluorescence were select by fluorescence-assisted cell sorting (FACS) and cloned in semi-solid medium. The analysis of the annexin V-FITC binding of the obtained clones showed that 10.7 % (± 0.26) of the population was able to bind annexin V before the stationary phase of culture growth (mid-log), as opposed to 2.11 % (±1.63) in wild type. The characterization of the cosmid DNA by nucleotide sequencing will allow the search, by comparison to genomic data-bases for possible candidate genes for the coding the responsible molecule for the phospholipid exposure. To confirm the role in the observed phenotype, the DNA fragments coding for the candidate genes will be cloned in a Leishmania expression vector and transfected into WT cells. The obtained clones are expected to bind annexin V in the log phase of culture growth as observed in the cells bearing the cosmids. Financial Support; FAPESP and CNPq.
- GENETICS, EVOLUTION AND TAXONOMY [430] P 082 - SPATIAL DISTRIBUTION AND THE INTRODUCTION OF LEISHMANIA INFANTUM GENOTYPES IN SÃO PAULO STATE, BRAZIL, INFERRED BY MULTILOCUS MICROSATELLITE TYPING DIRECTLY FROM INFECTED DOG TISSUES MOTOIE, G.1; FERREIRA, G.E.M.2; CUPOLILLO, E.2; CANAVEZ, F.3; MEIRA, C.S.1; PEREIRACHIOCCOLA, V.L.1 1.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.INSTITUTO OSWALDO CRUZ/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 3.GENOA BIOTECNOLOGIA SA, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania infantum; microsatellite; são paulo/brazil
Abstract: This study investigated the genetic characteristics of L. infantum population from São Paulo State (SP), Brazil, in order to collaborate with information about the possible origins of parasites and the introduction and spread of visceral leishmaniasis (VL) in this area. Multilocus microsatellite typing (MLMT) was performed using a set of 17 microsatellite markers. DNA was extracted from 250 tissue samples from dogs diagnosed with VL and 112 (45%) were genotyped: 67 from the northwest region (NWSP) and 29 from the southeast region (SESP) of SP. The results were correlated with 16 samples from Mato Grosso do Sul State (MS) (which borders NWSP). Although a small portion of samples was genotyped, it was possible to amplify multiple loci using DNA extracted directly from dog tissues, which generally present small amounts of Leishmania parasites. Despite the fact that MLMT defined 33 different genotypes, a low polymorphism was detected in the parasites studied with 10 loci being polymorphic. There are two main populations circulating in SP with strong genetic differentiation. POP-A was
composed by samples from SESP and NWSP and presented a weak signal of geographical substructure. The majority (93.75%) of MS samples belonged to POP-B, with just one sample in POP-A. POP-B was also comprised by 10.34% of SESP and 26.87% of NWSP samples. POP-A was composed by 73.13% of NWSP, 89.66% of SESP samples, and one sample from MS. The MLMT analysis supported the idea of canine VL being introduced in the NWSP by the traffic of humans and dogs from MS. In SESP occurred an introduction of a new L. infantum population. Probably the transmission was spread by traffic of infected dogs from other Brazilian regions or by introduction of imported dogs from other countries. All these data together contributed to the detection of the genetic profile of L. infantum populations in SP State.
- CELLULAR AND MOLECULAR BIOLOGY [431] P 083 - CLONING AND SEQUENCING OF ECTO - NTPDASE FROM LEISHMANIA AMAZONENSIS. CONDELO, H.S.1; RIBEIRO-GUIMARÃES, M.L.2; MOREIRA, O.C.2 1.IFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania amazonensis; ecto-ntpdases; virulence
Abstract: Leishmaniasis is a disease caused by parasites of the genus Leishmania, with approximately 12 million cases in the world (WHO, 2011). The forms of the disease in humans are cutaneous, mucocutaneous and visceral leishmaniasis. Leishmania amazonensis is the etiological agent of mucocutaneous leishmaniasis, which is characterized by a decrease in the immune response of the infected patient. Ecto-NTPDases are enzymes located at the external cell surface, that hydrolyze tri- and/or di-phosphate nucleotides, such as ATP and ADP, being essential for the acquisition of purines by trypanosomatids. Recent studies have shown a close relationship between the activity of ecto-and NTPDases infectivity of trypanosomatids. Thus, in this work, we aimed to perform the identification and molecular characterization of the ecto-NTPDase gene in L. amazonensis. This characterization can contribute to the understanding the role of this ecto-enzyme in the infectivity and virulence of the parasite. To develop this work, PCRs were performed using a set of degenerated primers based on the conserved regions of apyrases (ACRs) 1 and 5, aligned from the available DNA sequences of parasites from the genus Leishmania. After the DNA sequencing of the region comprised between the ACRs 1 – 5, we designed two new set of primers, to amplify the initial and terminal portions of the ectoNTPDase CDS. The PCR products were cloned and ten clones of each target were isolated and cultivated to proceed the plasmid purification, using the Kit TOPO TA. The nucleotide sequence was determined by DNA sequencing using the Genome Sequencing Platform of DNA (PDTISFiocruz), and analyzed using the MEGA software. The complete sequence was aligned with ecto-NTPDase mRNA sequences from other especies from the genus Leishmania, presenting high homology rates (92% with L. infantum, 91% with L. major and 83% with L. braziliensis). From this sequence we are now designing a set of primers to perform a quantitative Real-Time PCR assay, in order to compare the ecto-NTPDase gene expression among virulents and avirulents strains of L. amazonensis. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [432] P 084 - INTERLEUKIN 17 PRODUCTION AMONG BLOOD DONORS WITH ASYMPTOMATIC LEISHMANIA INFANTUM INFECTION AND CUTANEOUS AND VISCERAL LEISHMANIASIS INIESTA, L.1; FISA, R.1; JIMENEZ-MARCO, T.2; GIRONA-LLOBERA, E.2; GUILLEN, C.1; SEDEÑO, M.2; RIERA, C.1
1.DEPT MICROBIOLOGIA I PARASITOLOGIA SANITÀRIES, FACULTAT DE FARMÀCIA, UNIVERSITAT DE BARCELONA, BARCELONA, SPAIN; 2.FUNDACIÓ BANC DE SANG I TEIXIT DE LES ILLES BALEARS, PALMA DE MALLORCA, SPAIN.
Keyword:cytokine ; il17; asymptomatic infection
Abstract: Leishmania infantum causes leishmaniasis in the Mediterranean area with a diversity of the clinical manifestations that depends on the species and the immune response of the host, according to the associated cytokine profiles. The mechanisms by which individuals with subclinical L.infantum infection achieve control over the infection are as yet not understood. IL17 plays a critical role in inflammation and autoimmunity and participates in defence mechanisms against certain pathogens. A few studies have assessed the role of IL‐17 in parasitic diseases in humans and shown that IL-17 may protect against VL. In this study, we evaluated the cytokine profile, in asymptomatic blood donors (BD) and in patients with cutaneous (CL) and visceral leishmaniasis (VL) evaluating whether IL-17 production was associated with control of infection by L. infantum. Sera samples from: a) 39 BD from Balearic Islands (Spain) with asymptomatic leishmaniasis; b) 22 patients with CL; c) 6 patients with VL from Catalonia (Spain) and d) 32 negative control cohort, from Balearic Islands were tested. The asymptomatic L. infantum infection on BD were determined by Western blot (WB) and the presence of parasite in blood by a real-time PCR (RT-PCR). Diagnosis of CL and VL were confirmed by observation and/or isolation of parasite in tissue samples. The Human Th1/Th2/Th17 cytokine kit (Becton Dickinson and Company, BD Cytometric Bead Array (CBA)) was applied to measure production of IFN-γ, IL-2, TNF-α, IL-4, IL-6, IL-10 and IL-17. Data of this preliminary study shows that IL-17 was the cytokine with higher values in all populations studied and levels were higher in CL patients and in BD, suggesting its role in the control of the infection in these subjects. All populations showed level of IL-6, higher in VL patient’s samples and lowest levels of IL-6 in asymptomatic BD and in CL patients. It could be related to the dissemination of the disease and its role as suppressor of macrophages (pAs veterans of infection, Leishmania guyanensis parasites have been plaguing humankind for centuries, provoking a deleterious hyper-inflammatory immune response, destroying host tissue and forming the ulcerating lesions, which typify most forms of the disease. About 15% of patients develop secondary lesions in the mouth and nose, where parasites metastasise to mucocutaneous tissues creating corrosive and exceptionally disfiguring inflammation. Our lab has recently linked disease severity in these infections to a virus naturally residing within the cytoplasm of some Leishmania parasites. Here, LeishmaniaRNA-virus (LRV) can act as an independently immunogenic entity, where its RNA-based nucleic acidacts as a potent innate immunogen, triggering a destructive hyper-inflammatory cascade through Toll-Like-Receptor 3 recognition. Using Leishmania guyanensis clones, which are either naturally infected by LRV (V+) or depleted in it (V-), we set out to characterise the perpetrators of this chronic hyper-inflammation in a murine model of infection. Here, we found that V+ parasites potently induced the production of IL-17A as compared to their Vequivalents, thus insinuating that this pro-inflammatory cytokine plays a destructive role in the devolution and severity of metastatic leishmaniasis. Although IL-17 has a promiscuous role in immunity, stimulating both the innate and adaptive immune systems (as well as a plethora of non-immune cells), a major shared goal of these pathways is their self-propagation, where downstream effects converge to create a hyper-inflammatory feedback loop. Indeed, the Th17 T-cell population has been thoroughly vilified as the architect of many chronic and destructive inflammatory processes. Determining the role of IL-17 in LRV-based virulence is essential to our understanding of its pathogenesis and would stand to guide and justify a much-needed immunotherapeutic revolution in the treatment of complicated leishmaniases. - CELLULAR AND MOLECULAR BIOLOGY [604] P 116 - LEISHMANIA AETHIOPICA FIELD ISOLATES WITH AN IMMUNOGENIC LEISHMANIA RNA VIRUS FASEL, N.1; ZANGGER, H.1; DESPONDS, C.1; LYE, L.2; HAILU, A.3; BEVERLEY, S.M.2
1.UNIVERSITY OF LAUSANNE, EPALINGES, SWITZERLAND; 2.WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, UNITED STATES; 3.ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA.
Keyword:leishmania virus; inflammation; tlr-3
Abstract: Leishmania RNA virus (LRV) is a double stranded RNA virus which has been detected in Leishmania (Viannia) braziliensis and L. guyanensis species, which can cause not only cutaneous but also mucocutaneous (MCL) and disseminated (DCL) leishmaniases. This virus is composed of a capsid protein, a RNA dependent RNA polymerase (RDRP) and of a dsRNA genome of 5.3kb. In a mouse model, we showed that the viral dsRNA genome in L. guyanensis parasites is recognized by the host endosomal Toll-like receptor 3 (TLR3) and induced proinflammatory cytokines and chemokines. These TLR3-dependent immune responses render mice more susceptible to infection, and the animals develop an increased footpad swelling and parasitemia. MCL and DCL have also been described in other part of the world, e.g. in Ethiopia. We detected naturally occurring Leishmania RNA virus within some of L. aethiopica parasites isolated from patients. Three Lae-LRV genomes were sequenced from independent isolates confirming that LRV in L. aethiopica (Lae-LRV) belongs to the same Totiviridae family of LRVs found in South America species and present in a single isolate of L. major. Lae-LRV genomic organization is similar but not identical to the other LRVs. While LRV1 from L. braziliensis and L. guyanensis displays a +1 difference in the capsid/RDRP reading frame, and LRV2 of L. major capsid/RDRP polypeptides are encoded in the same frame, Lae-LRV genome displays a -1 difference in the capsid/RDRP reading frame. Similarly to L. guyanensis LRV, the presence of Lae-LRV in L. aethiopica induced a TLR-3 inflammatory response in L. aethiopica infected bone marrow macrophages. The presence of LRV and its detection could be a crucial step towards the development of new diagnostics and treatments for L. aethiopica infected patients. - CLINICAL LEISHMANIASIS [607] P 117 - ADULT VISCERAL LEISHMANIASIS IN TURKEY: A REPORT OF 16 CASES PULLUKCU, H.1; TURGAY, N.2; ISIKGOZ TASBAKAN, M.1; SIPAHI, O.R.1; UNVER, A.2; YAMAZHAN, T.1; OZENSOY TOZ, S.2; OZBEL, Y.2
1.EGE UNIVERSITY MEDICAL SCHOOL DEPARTMENT OF INFECTIOUS DISEASES AND CLINICAL MICROBIOLOGY, IZMIR, TURKEY; 2.EGE UNIVERSITY MEDICAL SCHOOL DEPARTMENT OF PARASITOLOGY, IZMIR, TURKEY.
Keyword:leishmaniasis; visceral; adult
Abstract: Leishmaniasis, both visceral and cutaneous, is public health problem in Turkey. The official number of visceral leishmaniasis cases (VL) is around 50 per year. In the present retrospective study, 16 VL cases diagnosed by detecting Leishmania amastigotes in bone marrow smears in Ege University Hospital between 2007 and 2012 were evaluated. Eleven and five patients were male and female, respectively. Their mean age was 46.43 ± 16.62 (min: 23; max: 74). All patients had fever and splenomegaly and hepatomegaly/anemia, leukopenia, pancytopenia and thrombocytopenia were observed in 15, 12, 11 and 10 patients respectively. All patients had a history of admission to several hospitals before etiological diagnosis and 13 of them had been diagnosed as hematological malignities, urinary system infections and influenza. Three patients were sent to university hospital for further analyses for the etiology of unknown fever. There were accompanying diseases in 7 patients as cyrosis, haematological malignancy, tuberculosis, and chronic renal failure which lead to a confusion in clinical diagnosis. Thirteen patients were treated with liposomal amphotericin B while Glucantime was administered to two patients. One patient with pulmonary tuberculosis was discharged from the hospital without treatment of VL. Three patients were died related to other causes and one patient was lost because of bleeding as a complication of VL.
Although, VL has been mainly seen in childhood ages in the Mediterranean Basin, adults who have chronic systemic diseases are always at risk in endemic regions and VL should be considered in differential diagnosis.
- CLINICAL AND EXPERIMENTAL IMMUNOLOGY [611] P 118 - LEISHMANIA CHAGASI DEATH TRIGGERED BY INHIBITION OF NEUTROPHIL APOPTOSIS PRATES, D.B.1; ANDRADE, M.B.1; FARIAS-LUZ, N.1; BARRAL-NETTO, M.1; BARRAL, A.1; BOZZA, M.2; BORGES, V.M.1
1.UNIVERSIDADE FEDERAL DA BAHIA/ CPQGM (FIOCRUZ), SALVADOR, BA, BRAZIL; 2.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmania chagasi; neutrophil; cell death
Abstract: Previously, we have demonstrated that Leishmania chagasi, the etiological agent of Visceral Leishmaniasis, causes the induction of neutrophil apoptosis. These cells, the neutrophils, are among the host’s first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Because different cell death pathways have been implicated in the pathogen killing or survival by host cells, we tested here whether inhibition of neutrophil apoptosis by pre-treatment with zVAD-fmk or zIETD-fmk increases L. chagasi killing, since it has been demonstrated that inhibition of caspase 8 reorients the cell death to necroptosis. Mouse peritoneal neutrophils obtained by thioglycolate injection were pre-treated with zVAD-fmk (a pan caspase inhibitor) or with appropriated controls and were infected with L. chagasi. Neutrophils pre-treated with zVAD presented a significant decrease on viable parasite (4.68x104 ± 1.59) compared with controls (cell culture medium: 13.06x104 ± 2.08; DMSO: 13.81 x104 ± 1.28 or zFA-fmk: 16.13 x104 ± 2.01). Interestingly, when we use a specific caspase 8 inhibitor, zIETD-fmk, we also observed a significant increase on parasite killing by neutrophils (cell culture medium: 13.06x104 ± 2.08; DMSO: 13.81 x104 ± 1.28; zIETD-fmk: 16.13 x104 ± 2.01). The inhibition of caspases increased the activation state of infected neutrophils. The neutrophils pre-treated with zVAD-fmk had higher concentrations of TNF-α (-zVAD 231.8pg/ml ± 88.3; +zVAD 474.5pg/ml ± 198.5) and ROS (MFI: -zVAD 44.95 ± 2.05; +zVAD 64.8 ± 1.27). In another hand, TGF-β release was reduced by this treatment (zVAD 107ng/ml ± 23.52; +zVAD 57.15ng/ml ± 12.15). Taken together, our data suggest that inhibition of neutrophil apoptosis by using caspase inhibitors have biological significance in the control of Leishmania infection, probably by inducing an inflammatory cell death type. Financial support: CNPq, INCT/iii
- GENETICS, EVOLUTION AND TAXONOMY [614] P 119 - POLYMORPHISMS AND AMBIGUOUS SITES PRESENT IN DNA SEQUENCES OF LEISHMANIA CLONES: LOOKING CLOSER OLIVEIRA, T.S.; BOITÉ, M.C.; FERREIRA, G.E.M.; TRANNIN, M.A.; SANTOS, B.N.; CUPOLILLO, E. FIOCRUZ - LABORATÓRIO DE PESQUISA EM LEISHMANIOSE, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:recombination; heterozygous; polyclonality
Abstract: Occurrence of polyclonality/infrapopulation in Leishmania might interfere in genetic variability analysis and parasite characterization. Ambiguous sites in DNA sequences may represent polyclonal populations, heterozygous or even “partially” heterozygous - considering chromosomal mosaicism. Aiming to look closer to these sites, six L. (Viannia) strains presenting ambiguous sites in previous 6PGD sequences were biologically (BC) and molecularly cloned (MC). 6PGD DNA sequences were obtained from new non-cloned cultures (NCC), as well as from BC and MC; the chromatogram was manually tracked for ambiguous sites and sequences were compared. Recombination analysis was performed in RDP software including other 120 L. (Viannia) sequences. The results showed that (i) three strains did not maintain the ambiguous sites in BC; two of these strains had identical sequences for all BC, suggesting monoclonal population. One showed a bi-alelic polymorphic site among BC, suggesting polyclonal population. The observation of ambiguous sites loss associated to identical BC might be due to asymmetric chromosome allotments during axenic culture, leading to the loss of heterozygosis, and clonal propagation of the new chromosomal arrangement; the polymorphisms observed can be explained by point mutations generating polyclonal population; (ii) Two strains (one with hybrid profile in 6PGDH isoenzyme) had identical BC which maintained the ambiguous sites observed in NCC and presented the possible alleles in the MC, as a heterozygous monoclonal; (iii) One strain maintained in the NCC the three ambiguous sites detected previously, but just one of these sites was still present in all BC. In the position of the two other ambiguous sites, the BC presented either the ambiguous site or one of the possible alleles. This case represents a polyclonal and heterozygous strain; (iv) After Recombination Detection Program (RDP), a L. braziliensis NCC strain was detected as a recombinant, and one BC of L. braziliensis and one MC of the L. naiffi/L. lainsoni – (hybrid profile in 6PGDH isoenzyme) were pointed as major and minor parental, respectively (P6 fold decrease in PMM susceptibility at amastigote stage of PMM-R (meanIC50±SD=61±7µM) compared to that of PMM-S (meanIC50±SD=11±1µM), while susceptibility to sodium antimony gluconate or miltefosine remained unaltered. The PMM-R parasite showed increased (2.5 fold) membrane fluidity, and decreased (3 fold) intracellular PMM accumulation as compared to PMM-S. Analysis of gene expression by real time PCR revealed an increased expression of ABC transporters; MDR1 (8.95±6.08 fold) as well as MRPA (11.47±0.22 fold) and protein phosphatase 2A gene (4.44±0.71 fold) in PMM-R parasite, evincing increased efflux of PMM. Full length gene sequencing of L. donovani rRNA gene failed to detect any mutations in the PMM-R strain of L donovani, unlike the reported observation in PMM resistant E. coli. Additionally, we evaluated the tolerance of PMM-R parasite towards nitrosative, oxidative and complement mediated stresses. PMM-R strain was more tolerant to nitrosative stress induced by SIN-1(above 2 fold) and SNAP (9 fold) as compared to PMM-S isolate, however, the tolerance to oxidative stress remained unaltered. The PMM-R parasites showed significantly higher resistance to complement mediated lysis. Macrophages infected with PMM-R parasites elicited higher (1.8 fold) IL-10 levels in comparison with PMM-S, indicating a better survival capacity of PMM-R. The study shed light on the mechanism of PMM resistance and fitness levels of PMM-R Leishmania donovani. This work was carried out under European Commission funded Kaladrug-R project (EC-FP7222895).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [182] P 364 - DECLINE IN IN VITRO DRUG SUSCEPTIBILITY OF CLINICAL ISOLATES OF LEISHMANIA DONOVANI FROM MILTEFOSINE TREATED CASES OF VISCERAL LEISHMANIASIS AND POST KALA-AZAR DERMAL LEISHMANIASIS (PKDL) BHANDARI, V.1; KULSHRESTHA, A.1; DEEP, D.K.1; STARK, O.2; PRAJAPATI, V.K.3; RAMESH, V.4; SINGH, R.1; SUNDAR, S.3; SCHONIAN, G.2; DUJARDIN, J.C.5; SALOTRA, P.1 1.NATIONAL INSTITUTE OF PATHOLOGY, NEW DELHI, INDIA; 2.INSTITUTE OF MICROBIOLOGY AND HYGIENE, BERLIN, GERMANY; 3.INSTITUTE OF MEDICAL SCIENCES, VARANASI, INDIA; 4.DEPARTMENT OF DERMATOLOGY, NEW DELHI, INDIA; 5.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM.
Keyword:visceral leishmaniasis; post kala azar dermal leishmaniasis; miltefosine
Abstract: With widespread resistance to antimonials in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy for visceral leishmaniasis (VL), however decline in treatment efficacy is reported. MIL has also been successfully used for treatment of post kalaazar dermal leishmaniasis (PKDL), a sequel of VL that constitutes an important parasite reservoir. In a set of VL/PKDL cases recruited for MIL treatment, we determined the in vitro susceptibility of L. donovani parasites obtained pre- and post-treatment at amastigote stage. MIL susceptibility of post-treatment isolates from cured VL cases (mean IC50±SD=2.43±1.44µM), was comparable (p>0.05) to that of the pre-treatment group (mean IC50 =1.86±0.75µM), while
that from relapses (mean IC50=4.72±1.99µM) was significantly lower (p=0.04). Similarly, PKDL isolates from relapse cases exhibited significantly higher (p=0.03) MIL tolerance compared to pre-treatment isolates (IC50=16.13±2.64µM and 8.63±0.94µM, respectively). Interestingly, PKDL isolates were more tolerant towards MIL than VL isolates. The inter-stage MIL susceptibility correlated significantly for parasite isolated from VL (r=0.70, p=0.0018); PKDL (r=0.78, p=0.046) and MIL induced parasites with high MIL tolerance (r=0.92, p=0.0001) when macrophage based amastigote assay and high-throughput resazurin-based promastigote assay were compared. The data suggests that the resazurin assay with promastigotes is applicable as a simplified biological tool for high throughput MILsusceptibility monitoring in clinical isolates of VL. Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes as well as changes in the mRNA expression of these genes that previously correlated with experimental resistance to MIL, were not evident in the clinical isolates. This work was carried out under European Commission funded Kaladrug-R project (EC-FP7222895).
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [183] P 365 - MECHANISM OF RESISTANCE IN FLAVONE RESISTANT LEISHMANIA DONOVANI TO CIRCUMVENT THE FLAVONE-MEDIATED CELL DEATH: ROAD TOWARDS PHENOTYPIC TO GENOTYPIC GAIN OF FUNCTION CHOWDHURY, S.; MAJUMDER, H.K. CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY, KOLKATA, INDIA.
Keyword:induced resistance; transporter; topoisomerase
Abstract: In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or LdMrp2. The localization is primarily in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be removed by treatment of macrophages with BLN. Amastigotes resistance is concommitant to the overexpression of macrophage multidrug resistance protein 2 (MRP2) transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element (ARE)-1 is activated followed by infection. The expression of this phase II detoxifying gene is regulated by Nrf2 (NFE2 – related factor 2) mediated Antioxidant Response Element (ARE) activation. In view of the fact that the signalling pathway of Phospho Inositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with intricate pathway of PI3-kinase–mediated nuclear translocation of Nrf2 which activates Mrp2 expression in macrophages. In contrast, phalloidin, an agent that prevents actin filaments from depolymerization, inhibited Nrf2 translocation and Mrp2 activation by pB25R infection. Parasites exposed to higher drug concentration (75 µM) show no difference in drug accumulation or efflux. Earlier it was found that flavones interact with LdTOP1LS to abrogate DNA transaction mediated life processes. Scanning of LdTOP1LS gene revealed that there are some mutations near to the active site of the enzyme. To our surprise this mutatnt enzyme is cross resistant to certain levels of other relative flavones like luteolin and quercetin. Taken together, these results provide an insight into the mechanisms by
which resistant clinical isolates of Leishmania donovani induces intracellular events relevant to drug resistance. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [184] P 366 - DETECTION OF LEISHMANIAL ANTIGENS IN PATIENT URINE BY QUANTITATIVE ELISA AND ITS POTENTIAL AS A TEST-OF-CURE ALBERTINI, A.1; HOMMEL, M.; ELLIS, S.2; HAQUE, R.3; HAQUE BHUIYAN, A.T.R.3; RAHMAN, R.4; FAIZ, M.A.5; MONDAL, D.3; CHILDERSTONE, M.6; PURCELL, N.6; NDUNG'U, J.1; PERKINS, M.D.1 1.FIND, GENEVA, SWITZERLAND; 2.DNDI, GENEVA, SWITZERLAND; 3.ICCDDR,B, DHAKA, BANGLADESH; 4.SHAHEED SUHRAWARDY MEDICAL COLLEGE AND HOSPITAL, DHAKA, BANGLADESH; 5.MALARIA RESEARCH GROUP, CHITTAGONG MEDICAL COLLEGE, DHAKA, BANGLADESH; 6.KALON BIOLOGICAL, GUILFORD, UNITED KINGDOM.
Keyword:diagnostics; antigen detection; vl
Abstract: Early identification of treatment failure in visceral leishmaniasis (VL) patients is critical to the successful control and elimination of the disease. An assay that could do this would also have potential as a pharmacodynamic marker. Both test indications would require a sensitive and quantitative assay. We report on the development of an antigen affinity purified (AAP) polyclonal double-antibody sandwich ELISA assay for leishmania antigens in urine, and its testing on patient and endemic control samples from South-East Asia. The AAP ELISA uses the same antigen as the KAtex latex agglutination test but is quantitative and does not require boiling of urine samples before testing. Urine samples were collected by DNDi in Bangladesh during a phase III/IV drug study, conducted at the Community Based Medical College (CBMC) of Mymenshingh, to determine the feasibility of implementing new treatment regimens for VL recommended by WHO (miltefosine (1.5-2.5 mg/kg in 1 or 2 doses/day) + paromomycin (11mg/kg/day) for 10 days, AmBisome® (5mg/kg) + miltefosine for 7 days, AmBisome® (5mg/kg) + paromomycin for 10 days, AmBisome® 15mg/kg) in a primary healthcare setting. The samples were obtained from parasitologically confirmed VL patients (collected at pretreatment, and on days 7, 15, 45 and 180 after initiation of treatment) and from healthy endemic controls. When a cut-off of 1 UAU/ml (arbituary units of urinary antigen per ml) was used, the AAP ELISA prototype was positive in 95.3% (102/107) of leishmania patient samples before treatment (baseline), while all the endemic controls (48/48) were negative. During the first 45 days after initiation of treatment, the level of leishmania antigen in urine decreased by 95% in majority of the patients. However, the cut-off of 1 UAU/ml still classified more than 50% of the samples as positive 45 days after initiation of treatment. While this assay has good potential as a test of cure, the appropriate time for testing after treatment needs to be defined, in line with patient follow-up visits. Further testing of this non-invasive prototype assay on samples from Eastern Africa is planned, before large-scale evaluation in the field. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [188] P 367 - EXPERIMENTAL STUDIES OF LIPOSOMAL BUPARVAQUONE USING DIFFERENT ROUTES OF ADMINISTRATION AND REGIMENS AGAINST LEISHMANIA (L.) INFANTUM DA COSTA SILVA, T.A.1; GARCIA-PEREZ, A.2; DEL VALLE, S.G.2; KESSLER, A.2; TEMPONE, A.G.1 1.DEPARTMENT OF PARASITOLOGY INSTITUTO ALDOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.GSK DISEASES OF THE DEVELOPING WORLD, TRES CANTOS, SPAIN.
Keyword:buparvaquone; liposomes; leishmania (l.) infantum
Abstract: Buparvaquone is a veterinary drug used to treat Theileriosis and has shown promising activities against protozoan parasites, including Leishmania spp. Despite its in vitro anti-leishmanial activity, in vivo experiments with L. (L.) donovani showed only a weak suppression of the parasite burden. Our previous studies demonstrated that intraperitoneal buparvaquone entrapped in phosphatidylserine-liposomes (BPQ-PS-LP) resulted in a significant suppression of Leishmania (L.) infantum in a hamster model. In this study the effectiveness of BPQ-PS-LP was evaluated at 0.4 mg/kg/day in a L. (L.) infantum-hamster model (n= 5/group) using different routes of administration (subcutaneous, intramuscular and intravenous) and regimens (5 and 10 consecutive days). The treatment was quantified by real-time PCR, using the detection of living amastigotes (RNA) in the spleen and the liver. BPQ-PS-LP showed the most significant reduction of the parasite burden when administered by subcutaneous route, reducing the amastigotes in spleen and liver by 98% (p0,48 milliseconds and refractory emesis associated with mild Amylase elevation. Only increase ALT/AST levels were frequent in the SSG group (p0,001) as well as a prolonged period with symptoms in those from the SSG group (p=0,012). Subjects older than 25 years had higher chances to present those symptoms [OR: 2,45; p=0,023 (CI:1.13-5.33)].Our findings show that efficacy of MA and SSG was similar and only ALT/AST elevations, dysgeusia, dizziness and headache were more frequent during SSG treatment. Age was an important determinant of laboratorial and clinical side effects. Partially funded (statistical analysis) by Sanofi. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [928] P 536 - DIAGNOSIS OF URBAN VISCERAL LEISHMANIASIS IN NEIVA AYALA, M.S. INSTITUTO NACIONAL DE SALUD, BOGOTA, COLOMBIA.
Keyword:visceral leishmaniasis; visceral leishmaniasis; visceral leishmaniasis
Abstract: INTRODUCTION: Visceral leishmaniasis (VL) is one of the world's neglected diseases. In Colombia, mainly affects children under 15 years and can be potentially fatal without prompt treatment. Diagnostic confirmation of cases is done by direct observation of the parasite in samples of tissue smears obtained by splenic aspiration with a sensitivity of 96 to 98%, 70% in bone marrow and 58% in lymph nodes and by quantifying IgG antibodies by indirect immunofluorescence. According to the guidelines established in the event monitoring protocol, serological tests are considered useful in the diagnosis and monitoring of disease. MATERIALS AND METHODS: To 7 children under 3 years who met the probable case definition of LV, prolonged fever, and one or more of the following symptoms: hepatosplenomegaly, anemia, thrombocytopenia, and repeated infections, underwent direct and serological diagnosis by indirect immunofluorescence (IIF), technique developed in the Parasitology Reference Laboratory of the National Institute of Health (NIH) and made from strains of L. infantum circulating in the country that were isolated, cultured and identified. RESULTS: One of the children was positive for antileishmanial IgG antibodies (14.3%) versus 85.7% (6 children) positive by direct examination obtained by : splenic aspirate (12.5%) and 87.5 % by bone marrow aspiration, which is the most used test for medical staff due to security reasons its execution and lower risks to the patient. DISCUSSION: Given the high lethality of LV is essential to diagnose and provide timely care to prevent the occurrence of severe or fatal infections. According to the results, the low reactivity in the immune response of patients in critical clinical condition and the high sensitivity of direct examination suggests implementing the practice of direct diagnostic test in endemic areas of the country and strengthen the skills of medical personnel to direct sampling of spleen due to the preferential localization of parasites in this organ.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL
[930] P 537 - PRODUCTION OF RECOMBINANT CHIMERIC PROTEINS FOR VACCINE AND DIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS FARIA, A.R.; DAMASCENO, L.M.; GAZZINELLI, R.T.; ANDRADE, H.M. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:diagnosis; recombinant; protein
Abstract: Visceral Leishmaniasis (VL) is a neglected parasitic disease, widely distributed and with increasing incidence in urban and peri-urban areas. The dogs act as disease reservoirs, when it is caused by Leishmania infantum chagasi. Since the control of canine disease is managed by the diagnosis and subsequent euthanasia of infected dogs, there is a need for reliable diagnostic tests. Moreover, the development of a vaccine that allows protection against the infection it is also necessary. To contribute to disease control, the goal of this work is the production and the characterization of two chimeric recombinant proteins, which will be tested for LVC diagnostic and vaccine. To be used in the diagnosis, a chimeric protein was constructed with ten peptides previously identified and tested in ELISA with sensitivity and specificity around 80% and accuracy (AUC) of 0.9 (Faria et al, 2011). These 10 peptides were included in a synthetic gene, which had been cloned and expressed in Escherichia coli. After purified, it has been used in ELISA and in the development of an immunochromatographic assay. Sensitivity and specificity of 91.8% and 88.8%, respectively, and AUC=0.947, were achieved using this chimeric protein in ELISA, when testing canine sera (n=61 infected and n=10 uninfected ones). In parallel, another chimeric protein was produced, which will be used in dog immunization. For this, we used 20 peptides previously described as good T cell epitopes (Costa et al, 2011). In summary, our studies demonstrate that the use of chimeric proteins has great potential in diagnosis and immunization, what may benefit Canine Visceral Leishmaniasis control programs. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [932] P 538 - ENZYME INHIBITION VERSUS NITRIC OXIDE PRODUCTION BY BENZOPHENONE DERIVATIVES DE ALMEIDA, L.1; ALVES, K.F.1; MACIEL-REZENDE, C.M.1; PIRES, F.R.1; IZIDORO, M.A.2; DOS SANTOS, M.H.1; MARQUES, M.J.1 1.UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania; nitric oxide; cysteine protease
Abstract: Considered as neglected tropical diseases, leishmaniasis are caused by protozoa of the genus Leishmania. The drugs available for the treatment of this disease exhibit various side effects. New compounds classes, benzophenones, have been described for your activity on several human pathogens, including T. cruzi and Leishmania. The objective of this study was to try to find possible action mechanisms of benzophenone derivatives that have proven to be effective on both forms of Leishmania (L.) amazonensis, since this class of compounds have been showing effectiveness on several human pathogens. Therefore with the derivatives (LFQM-116, LFQM-117, LFQM-119, LFQM-120 and LFQM-121) was performed dosage of nitric oxide (NO) and evaluation of enzyme inhibition potential of cysteine proteases representatives (papain, cruzain). To assess the nitric oxide (NO) production in cell culture was used Griess reaction, which evaluates the production of nitrite, a degradation product of NO. Culture supernatants from murine peritoneal macrophages infected with L. (L.) amazonensis and in contact with the substances tested or LPS were collected after 48 hours. For testing enzyme inhibition, papain and cruzain were pre-activated with DTT. The benzophenone derivatives were
added and the dosage of enzyme activity was initiated by adding of substrate Z-FR-MCA in the reaction. Moreover, molecular docking was also performed using AutoDock Vina. It can be seen that not all derivatives induce macrophages to produce the equivalent amount of NO that is produced by cells in contact with LPS. The compounds LFQM-119, LFQM-120 and LFQM-121 (NO = 13.61, 13.01 and 11.44 µM, respectively) induced more the NO production than the other ones. To the papain’s inhibitory activity the best results were exhibited by derivatives LFQM116, LFQM-117 and LFQM -119 (IC50 values = 27.91, 42.78 and 48.20 µM, respectively). But LFQM-119 showed no Hydrogen bonds (H-bonds) interactions with the enzyme’s active site residues. The best results for cruzain’s inhibitory activity were showed by LFQM-119, followed by LFQM-116 and LFQM-117 (IC50 values = 28.13, 31.24 and 33.69 µM, respectively), additionally these two last ones exhibited H-bonds interactions with enzyme’s active site residues. At the end of these tests can be observed that the derivatives LFQM-117 and LFQM116 were the main inhibitors to the proteases evaluated. While derivatives LFQM-119, LFQM120 and LFQM-121 stimulated more the NO production. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [943] P 539 - CONJUNCTIVAL SWAB PCR AS A SPECIFIC AND SENSITIVE TOOL IN THE DIAGNOSIS OF CANINE LEISHMANIASIS USING GENERIC AND SPECIFIC PRIMERS OLIVEIRA, T.M.F.S.1; PEREIRA, V.F.2; BENASSI, J.C.3; DA SILVA, D.T.4; STARKE-BUZETTI, W.A.4 1.FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 2.FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 3.FACULDADE DE ZOOTECNIA E ENGENHARIA DA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL; 4.FACULDADE DE ENGENHARIA DA UNIVERSIDADE ESTADUAL PAULISTA, ILHA SOLTEIRA, SP, BRAZIL.
Keyword:conjuntival swab; dogs; pcr
Abstract: Vector-borne diseases, leishmaniasis are endemic in many countries, and an increasing problem in veterinary medicine and public health, due to their zoonotic importance. Parasites of the genus Leishmania are etiological agents of both, cutaneous and visceral forms of human and viscero-cutaneous canine leishmaniasis (CL). Leishmaniasis diagnose can be made by direct methods, such as smear prepared with different lymphoid organs and polymerase chain reaction (PCR), or by indirect methods such as enzyme immunoassay (ELISA) and Indirect Immunofluorescence (IFAT). Currently, molecular techniques, such as PCR, are being widely used for its sensitivity and specificity and it is known that PCR efficiency is directly associated to used primer and biological sample. Thus, the aim of the present study was diagnose CL using generic and specific primers with DNA samples from conjunctival swab (CS). CS is easy to collect and a noninvasive technique that has been shown to be a good biological sample to screen positive animals by PCR, even without clinical signs. To test and compare the use of CS samples by PCR analysis, CS and blood were collected from 213 dogs in an epidemiological survey in an endemic area from Brazil. The present study used three different pair of primers; two of them generic (L1/ L2 and 13A/13B) and one specific to L. infantum (MC). The CS PCR results showed 29 (61.8%) positive dogs using 13A/13B primers and 27 dogs (57.5%) using L1/L2. Specific primers MC1/MC2 showed 15 (32%) CS PCR positive dogs, demonstrating the possible presence of more than one Leishmania specie. Results of 13A and 13B primers were compared to blood PCR and IFAT from the same dogs. Using IFAT as gold standard, sensitivity and specificity of CS PCR were respectively 58.6% and 94.0%, positive predictive value 61.0%, negative predictive value 94.0%, and kappa index 0.53, which demonstrates moderate agreement between tests. PCR blood demonstrated a sensitivity of 24.1%, specificity 88.5%, positive predictive value 25% and negative predictive value of 88% and kappa index 0.13, which demonstrates a low agreement between tests. The results showed that conjunctival swab can be a good tool in diagnose CL since is easier to collect than blood or lymphoid organs and has a good sensitivity and specificity. The three screened primers were capable to detect parasite
DNA in CS, which can be useful for epidemiological studies and for direct diagnosis of canine visceral leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [948] P 540 - INTERPRETATION OF DIAGNOSTIC TEST PERFORMANCES TO IDENTIFY THE CANINE RESERVOIR OF VISCERAL LEISHMANIASIS: RELATIONSHIPS BETWEEN ANTI-LEISHMANIA ANTIBODY AND PARASITE LOADS USING FTA BLOOD SPOT TECHNIQUES. NUNES, C.M.1; BELOTI, C.C.1; CALVO-BADO, L.2; BONFIETTI, L.X.3; HIRAMOTO, R.M.3; COURTENAY, O.2 1.UNESP, UNIVERSIDADE ESTADUAL PAULISTA, FMVA, ARAÇATUBA, SP, BRAZIL; 2.UNIVERSITY OF WARWICK, COVENTRY, UNITED KINGDOM; 3.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL.
Keyword:canine visceral leishmaniasis; diagnosis; fta blood spot techniques
Abstract: Brazilian MoH recommendations for reservoir control against zoonotic visceral leishmaniasis transmission is based on identification of IgG antibody in canine sera samples. This study assesses the relative performances of the diagnostic kits currently adopted (DPP rapid test, and EIE ELISA Bio-Manguinhos®) compared to crude leishmania antigen ELISA and kDNA quantitative real-time PCR, the latter tested on FTA card blood spot samples for ease of collection. 400 characterised dogs from 28 endemic municipalities in São Paulo state were tested. Results are discussed in context of field logistics and laboratory expertise requirements. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [953] P 541 - IN VITRO LEISHMANICIDAL AND IMMUNOMODULATORY EFFECTS OF NEW THIOSEMICARBAZONES DERIVATIVES SILVA, A.C.1; SANTOS, T.A.R.1; GOMES, P.A.T.2; MOREIRA, D.R.M.2; LEITE, A.C.L.2; PEREIRA, V.R.A.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:leishmania amazonensis; synthetic compounds; nitric oxide
Abstract: Leishmaniasis is a chronic disease caused by kinetoplastid protozoa belonging to the genus Leishmania, which are transmitted from animals to humans by sandflies. Traditional drugs used in treatment are expensive, have many side effects and has been show patients nonresponsive because of drug-resistant parasite strains. So the need for the development of new, effective, cheap, and safe drugs for the treatment of leishmaniasis still very important. In this work was investigated the antileishmanial and immunomodulatory properties of five new molecules derived from thiosemicarbazone structure, respectively coded by PT01, PT01.2, PT01.3, PT01.4 and PT01.6. For this, Leishmania amazonensis promastigotes (106 parasites/mL) were incubated in the absence or presence of different concentrations (0,19 to 100 ug/mL) of molecules for 96h. After that, the parasite growth was determined by counting in a Neubauer chamber and the concentration that inhibited culture growth by 50% (IC50) was determined. Toxicity in mammalian cells was evaluated in J774 A.1 macrophages incubated or not with different concentrations (1 to 100ug/mL) of compounds for 48h, by MTT method. The 50% cytotoxicity concentration (CC50) was determinate. Selectivity Index (SI) was calculated as the ratio of CC50 to IC50. J774A.1 cells were incubated with the CC50 concentration for 48h and the nitric oxide (NO) levels were assayed in the supernatants by Griess reagent method. The PT01 compound showed the best result against Leishmania amazonensis with low IC50 (0.91 ug/mL) and high
CC50 (14.61ug/mL) presenting the highest SI: 16.05. The others molecules also demonstrated leishmanial activity with low IC50 values, between 1 and 5 ug/mL, and high SI, between 8 and 14. PT01, PT01.2 and PT01.4 stimulated significantly production of NO in macrophages. It is known the important role of NO in the phagocytosis of intracellular pathogens macrophage mediated and a new drug development which is able to act directly on the parasite and in the immune system stimulation, with low toxicity to host cells, can be an ideal approach to a novel treatment against leishmaniasis. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [954] P 542 - CUTANEOUS LEISHMANIASIS: COMPARATIVE STUDIES OF THE METHODS COMMONLY USED FOR DIAGNOSIS IN THE PRESENCE AND ABSENCE OF ANTIBIOTICS PASCUAL, Y.1; DELGADO, O.1; BORGES, R.2; RIVAS, M.1; ROSAS, J.1
1.SECCION DE INMUNOPARASITOLOGIA, INSTITUTO DE MEDICINA TROPICAL, UNIVERSIDAD CENTRAL DE VENEZUELA, CARACAS, VENEZUELA; 2.INSTITUTO DE BIOMEDICINA, CARACAS, VENEZUELA.
Keyword:scarification smears; intradermal test; indirect immunofluorescence assay
Abstract: Cutaneous leishmaniasis (CL) is a parasitic disease considered a public health problem worldwide, early and accurate diagnosis is required for an appropriate treatment. The presumptive diagnosis is based on epidemiological history and the clinical characteristics of lesions, which may have secondary infections, mostly bacterial. Confirmatory tests are applied, parasitological (smear, histopathology, culture and animal inoculation), cell-mediated immunity (intradermal test, IDT) and humoral (indirect immunofluorescence assay, IFA), enzyme-linked immunosorbent assay (ELISA) as well as recently the PCR use and the specific hybridization. The objective of this study was to determine the correlation between tests used in routine diagnosis of CL and if it changes with the use of antibiotics. A comparative study was performed in 114 patients attending in the Inmunoparasitología Section, with skin lesions suggestive of CL, during august/2009-september/2010. Scarification smears, IDT and IFA were performed to the patients. The data was analyzed by two statistical analysis programs SPSS v.10.0 and Microsoft Office Excel 2007. There was a good agreement (Kappa = 0.763) between scarification smears and IDT, weak between scarification smears and IFA (Kappa = 0.397) and moderate between IDT and IFA (kappa = 0.553), without the use of antibiotics. Concordance between scarification smears and IDR decreased antibiotic use (kappa = 0.491), while it was higher between scarification smears and IFA (kappa = 0.498) and between IDT and IFA (kappa = 0.948). In conclusion, the use of antibiotics improved the positivity rate of scarification smears and consequently modified the strength of agreement between it and the immunological tests. The use of antibiotics is recommended before taking samples and apply both tests to a comprehensive confirmatory diagnosis in patients with suspected clinical-epidemiological of cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [971] P 543 - ANTILEISHMANIAL ACTIVITY OF SELAGINELLA SELLOWII HIERON. (SELAGINELLACEAE) EXTRACT ON LEISHMANIA (LEISHMANIA) AMAZONENSIS AMASTIGOTES RIZK, Y.S.1; BOSQUIROLI, L.S.S.1; FISHER, A.1; DE ARAÚJO, V.C.P.1; DA COSTA, E.C.1; QUEIROZ, D.P.S.1; DE OLIVEIRA, A.N.1; MARQUES, M.C.S.2; CAROLLO, C.A.1; DE ARRUDA, C.C.P.1 1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CAMPO GRANDE, MS, BRAZIL; 2.UNIVERSIDADE CATÓLICA DOM BOSCO, CAMPO GRANDE, MS, BRAZIL.
Keyword:plant extracts; in vitro infection; cutaneous leishmaniasis
Abstract: Species of Pteridophytes of the genus Selaginella have been cited as having anti-cancer, antitrypanosomal and anti-leishmania activity. Widely found in the Pantanal of Mato Grosso do Sul, Brazil, Selaginella sellowii presents secondary metabolites with potential use in the treatment of American cutaneous leishmaniasis (ACL). This study evaluated the survival of Leishmania amazonensis amastigotes within peritoneal macrophages submitted to the action of acetone extract of S. sellowii, at concentrations of 50, 25 and 12.5 µg/mL, along 24, 48 and 72h. Untreated cells were used as control. The acetone extract (12.5 µg/mL) showed high activity against L. amazonensis amastigotes, reducing in 59,7% the infection index of infected macrophages after 72h. In the other concentrations it was not possible to estimate the infection index due to the insufficient number of cells, suggesting a possible toxicity of the extract in higher concentrations. Further studies are necessary to elucidate the cytotoxicity and characterization of the compounds responsible for the antileishmanial activity. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [975] P 544 - VALIDATION OF HIGH THROUGHPUT DIAGNOSTIC ASSAYS FOR EPIDEMIOLOGICAL SCREENING FOR LEISHMANIA CALVO-BADO, L.A.; KRAVAR-GARDE, L.; SUZANNE, G.; ORIN, C. THE UNIVERSITY OF WARWICK, COVENTRY, UNITED KINGDOM.
Keyword:qpcr; automated system; diagnostics
Abstract: Monitoring Leishmania exposure, infection and parasite loads in reservoir hosts for epidemiological evaluation involves technical, analytical and interpretational challenges. Selection of the appropriate diagnostic assay will also be detirmined by cost. This paper describes the validation of a high throughput diagnostic platform for quantification of Leishmania in buffycoat, dried blood spot-FTA cards, ear tissue and conjunctiva swab samples from endemic dog populations in Brazil and Greece. The DNA extraction, ELISA and PCR quantification was performed using a robotic arm. The sample types and diagnostic assays were compared for performance metrics, and discussed in relation to financial and logistic considerations. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [979] P 545 - FUROSEMIDE IS THERAPEUTICALLY EFFECTIVE AGAINST CUTANEOUS LEISHMANIASIS AND SHOWS INCREASED ACTIVITY AGAINST ANTIMONYRESISTANT LEISHMANIA DONOVANI PARASITES. COSTA, N.A.1; AMARAL, E.E.A.2; FERNANDES, J.R.M.1; BERGMANN, B.R.1 1.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 2.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:furosemide; resistance; leishmania
Abstract: Intramuscularly administered pentavalent antimonials such as Glucantime® and Pentostam® are the first line therapy against cutaneous leishmaniasis despite their toxicity and drug-resistance potential. Previously, we showed that furosemide, a clinically approved anti-hypertension drug, can also inhibit the Na+-ATPase activity of Leishmania amazonensis promastigotes (De Almeida-Amaral, E.E et al., 2008). In this work, we evaluated: 1) the oral antileishmanial activity in mice; 2) the responsiveness of antimony-resistant promastigotes of L. amazonensis
and L. donovani to furosemide; and 3) the correlation between Na+-ATPase expression and sensitivity of L. amazonensis and L. chagasi (syn. L.infantum) to furosemide. For in vivo efficacy, BALB/c mice were infected in the ear with L. amazonensis promastigotes. After 7 days, the animals were daily treated with 50 mg/kg of furosemide by the oral route for 79 days. Controls received 14 doses of 20 mg/Kg of Pentostam® or PBS. The lesion sizes were measured throughout the infection with a dial calliper. On day 86 of infection, the animals were sacrificed and the parasite load was quantified by Limiting Dilution Assay. The results showed that oral furosemide prevented lesion growth in a more pronounced way than i.p. Pentostam®. To test the sensitivity of Sb-resistant parasites to furosemide, L. amazonensis and L. donovani promastigotes were rendered resistant to antimony by successive culture with increasing concentrations of antimony tartrate (SbIII). The Sb-resistant and Sb-sensitive promastigotes were cultivated for 72h at 26°C in the presence of furosemide, and growth inhibition was evaluated by MTS. The results showed that contrary to L. amazonensis in which Sb-resistance induced cross-resistance to furosemide, Sb-resistant L. donovani was found to be even more sensitive to furosemide than wild-type parasites. Finally, we correlated the sensitivity to furosemide with the expression of Na+-ATPase in normal L. amazonensis and L. chagasi as measured by qRT-PCR. The results showed that L. chagasi promastigotes expressed more Na+ATPase mRNA than L. amazonensis. However, the sensitivity of L. chagasi to the inhibitory effect of furosemide was lower than L. amazonensis (IC50= 575 µM and 147 µM, respectively). Altogether, these results show that the antileishmanial activity of furosemide is extensive in vivo against cutaneous leishmaniasis in a manner apparently unrelated to its classical Na+-ATPase inhibitory effect, and although yet to be directly demonstrated, has a therapeutic potential in the treatment of Sb-resistant L. donovani infections. Financial support: CNPq. De Almeida-Amaral, E.E; Caruso-Neves, C; Pires, V.M.P.; Meyer-Fernandes, J.R. (2008) Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity. Experimental Parasitology, 118(2):165-171. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [983] P 546 - DIAGNOSIS AND TREATMENT PROCEDURES IN A CASE OF LEISHMANIASIS WITH EXTENSIVE MUCOUS INVOLVEMENT. SILVEIRA, A.M.1; CARDOSO, R.M.1; FREIRE, G.M.1; AQUINO, T.A.1; ROSELINO, A.M.F.2; GOMES, C.M.1; JANINO, M.P.D.1; DE PAULA, N.A.2; RIBEIRO SAMPAIO, R.N.1
1.UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL; 2.UNIVERSIDADE DE SÃO PAULO, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:mucous leishmaniasis; diagnosis; treatment
Abstract: The American tegumentary leishmaniasis (ATL) has a high incidence in Brazil and a potential to determinate disfiguring lesions. A case of mucosal leishmaniasis and it's diagnostic molecular procedures are reported in this study. A 58-year-old male patient, presented nasal obstruction and corysa for 2 years, without improvement with the use of nasal corticoid. Dermatologic exam detected redness, crusting and infiltration in nasal mucosa. The leishmanin skin reaction was positive (30mm) but indirect immunofluorescence wasn`t reactive. Videonasolaryngoscopy detected bulging of the posterior wall of the right oropharynx and hypopharynx, ulceration on the posterior wall of the oropharynx, granulomatous lesion of ventricular band and left vocal fold (VF). In addition, the exam found granulomatous lesions in floor and wall of nasal vestibule, with bilaterally narrowing of the nasal cavity. Histopathological examination showed intense inflammatory histiocytic infiltrate, sometimes forming nodules with giant cell permeation, suggestive of ATL. Polymerase chain reaction-restriction fragment length polymorphism with digestion by Hae III enzyme obtained by filter paper sample and nasal swab
of nasal mucosal detected L. (V.) braziliensis species. Hospitalization was required because of the evidence of severe edema in the VF, soft palate and nasal mucosa, for a course of oral corticosteroids and begining of pentavalent antimony treatment. Mucosal involvement in LTA is uncommon and results from haematogenous or lymphatic dissemination of amastigotes from the skin to the nasal-oropharyngeal mucosa. Oral mucosa and pharyngeal sites aren`t preferentially involved in ML. The lower temperature at the nose and areas of the bolus passage offer favorable condition to installation of the parasite. The presence of the lesion in VF associated with the use of medication without hospitalization could lead to the development of asphyxia, since this finding wouldn`t be discovered in the absence of videonasolaryngoscopic routine examination. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1002] P 547 - IN VITRO AND IN VIVO ACTIVITY OF QUERCETIN AND CHALCONE CH8 IN VISCERAL LEISHMANIASIS SILVA, C.I.M.; FALCÃO, C.A.B.; BERGMANN, B.R. FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:oral treatment; visceral leishmaniasis; flavonoids
Abstract: The activity of the flavonoids quercetin (QC) and chalcone CH8 in the oral treatment of cutaneous leishmaniasis has been investigated by our group in previous studies with successful results. With the aim to investigate whether such activity is extended to visceral leishmaniasis, we performed both in vitro and in vivo assays using the murine model of infection with Leishmania infantum (syn L. chagasi). Firstly, promastigotes of L. infantum were incubated with CH8 or quercetin, resulting in an IC50 of 15,4uM and 423,8uM respectively. To assess in vivo leishmanicidal activity, BALB/c mice were infected in the caudal vein with 2x107 promastigotes at stationary growth phase and, two days later, subjected to 28 daily doses of 150 mg/kg of CH8 or 16 mg/kg of QC administered by oral gavage. Animals were euthanized at the end of the treatment and the serum analyzed for quantification of the enzymes alanine aminotransferase, aspartate aminotransferase and creatinine related to hepatic, cardiac and renal toxicity respectively. Individual livers and spleens were collected for quantification of parasite burden by limiting dilution assay and in situ levels of oxide nitric and cytokines. Spleen cells were also tested for their oxide nitric and cytokine response to lipopolysaccharide and concanavalin in vitro. No signs of toxicity were found for treatment with either of the drugs. In terms of parasite burden, treatment with CH8 led to a significant reduction in both the liver and the spleen. Reduced levels of oxide nitric were found in the spleens of mice treated with CH8 which seems to be a result of their lower parasite burdens. Results also showed the efficacy of QC with a significant reduction of the parasite levels in the liver and a tendency for lower burdens in the spleen. QC appears to have led to a modulation of the host immunity as suggested by the significantly increased levels of IFN-γ and TNF-α found in the spleens of mice at the end of the treatment. Further evidence of QC’s immunostimulatory effect is the increased production of oxide nitric and IFN-γ by splenic cells stimulated in vitro, when compared to the untreated control group. Altogether, these results show the safe and effective application of CH8 and QC flavonoids in the treatment of visceral leishmaniasis, with the great advantage of using a noninvasive oral route. The mode of action of each drug appears to have a distinct nature, with the former one showing mainly a direct effect on the parasite, while the later appears to also promote the establishment of an effective immune response against the parasite in the murine host. Finantial support by Fundação para a Ciência e Tecnologia (FCT), Portugal. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1007]
P 548 - ARGINASE I, WHAT IS ITS ROLE IN LEISHMANIA BRAZILIENSIS INFECTION? SANTIAGO, R.C.; COSTA, J.F.; BORGES, V.M.; DE OLIVEIRA, C.I. CPQGM/FIOCRUZ, SALVADOR, BA, BRAZIL.
Keyword:arginase ; no; leishmania braziliensis
Abstract: Arginase is an enzyme that metabolizes arginine to ornithine. The increased availability of ornithine favors the synthesis of polyamines, which is important for parasite proliferation. This work aims to advance the knowledge of cutaneous leishmaniasis (CL) caused by Leishmania braziliensis(Lb), evaluating the role of arginaseI (arg1) and nitric oxide (NO), in lesion development and parasite persistence at secondary lymphoid organs. BALB/c mice wereinfected with Lb in the ear dermis.Lesion development, parasite load, arg1 activity and NO production in the ear and in draining lymph nodes (dLN)were evaluated as well as cytokine production. Infected animals develop an ulcerated lesion that heals spontaneously atthe second month of infectionaccompanied by parasite clearance. Parasites, however, persistin the dLN, until the last point evaluated - 6 months after infection. At the infection site, arg1 activity and NO production follow parasite replication and clearance whereas in dLNs, arg1 activity is associated with parasite persistence. Inhibition of arg1 during Lb infection, using Nor-NOHA, a competitive inhibitor, led to a significantdecrease in lesion size accompanied by a reduction in parasite load in the ear and in dLN. Interestingly, this outcome was associated with higher IL-4 and IL-10 production by dLN cells.Administration of arginine, the common substrate for enzymes iNOS and arginase, during the initial phase of Lb infection exacerbatedinjury and parasite replication. Collectively, we observed that arg1 activity and NO production parallel lesion development and healing at the infection site. At sites of parasite persistence, such as the dLN, arg1 activity is increased whereas NO production is suppressed. Our data suggest that arg1 maybe involved in parasite persistenceand, possibly, immunity to re-infection with L. braziliensis. Financial support: CNPq and FIOCRUZ
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1010] P 549 - IN VITRO ACTIVITY OF FRACTIONS AND CRUDE EXTRACTS OF LIBIDIBIA FERREA AGAINST LEISHMANIA AMAZONENSIS WYREPKOWSKI, C.C.1; SANTOS, P.A.2; SOUZA, G.O.2; SOARES, F.V.1; GRAFOV, A.3; GRAFOVA, I.3; LIPIK, V.4; FRANCO, A.R.1 1.INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA, MANAUS, AM, BRAZIL; 2.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS, AM, BRAZIL; 3.UNIVERSITY OF HELSINKI, HELSINKI, FINLAND; 4.NANYANG TECHNOLOGICAL UNIVERSITY, SINGAPORE, SINGAPORE.
Keyword:fabaceae; bioassay; natural products
Abstract: Leishmaniasis is an endemic disease in the Northern region of Brazil transmitted by a sand fly vector, when it takes a blood-meal on a mammal or human. In most cases the disease is revealed by cutaneous or muco-cutaneous lesions. Treatment of the cutaneous leishmaniasis is difficult due to a limited number of drugs capable to eliminate the intracelular parasite form. The drugs for conventional treatment (Glucantime® and Pentamidine) have to be injected either into a large muscle or intravenously. However, they might have various adverse effects such as: malaise, myalgia, headaches; changes in liver, kidney, and pancreas. Vegetal extracts from native Amazonian species constitute a promising source of natural bioactive products for
alternative treatment of leishmaniasis. Particularly, the genus Libidibia has become an object of detailed chemical and biological investigations. Branches, leaves and fruits of L. ferrea were collected in Manaus (AM, Brazil). The plant material was dried, grinded, and the extracts were prepared with hexane and methanol using sonication treatment for 20 minutes each, and partitioned between dichloromethane (DCM), ethyl acetate, and n-butanol. This fractions and crude extracts were filtred in milipore 0.22 µm, dissolved in complete Schneider Insect Medium, and tested in bioassay using microplates against promastigotes (106cells/mL) of Leishmania amazonensis in different concentrations to determine CI50. Pentamidine was used as the positive control, DMSO and the extraction solvent were negative controls. The number of surviving promastigotes were counted in a Neubauer’s chamber, with Trypan Blue, and compared with the controls after incubation for 24 and 48 hours at 24.9 oC. All tests were performed in triplicate. The most effective fraction (CI50< 62 µg/mL) was obtained by DCM treatment of the methanolic epicarp extract. Hexane crude extract of leaves (CI50< 125 µg/mL) showed a moderate activity, when compared to literature data on other natural plant extracts. The parasites grown in presence of the above samples and transferred into a new cultivation medium without extracts did not show any viable cell. Hence, the extracts possess a pronounced leishmanicidal activity, but the mode of action has to be an object of further investigation. Chemical studies of the samples provide additional information for understanding of the composition and the mechanism of action of the natural products in question. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1013] P 550 - IN SILICO SCREENING OF PROTEINASES INHIBITORS FOR POTENTIAL DRUGS FOR TREATMENT OF LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTION. SILVA, F.S.; ALMEIDA, M.S.; RIBEIRO, M.L.; SOUZA, R.S.; PEREIRA, B.S.; CAFFARENA, E.R.; ALVES, C.R. FUNDAÇÃO OSWALDO CRUZ, RIO DE JANIRO, RJ, BRAZIL.
Keyword:l. (v.) braziliensis; proteinases; isoforms
Abstract: Currently, the standard screening of compounds to inhibit Leishmania sp. proteinases does not usually consider the following points: (i) the possibility of the compound targeting multiple proteinase classes, (ii) the possible existence of different isoforms of the target proteinase, and (iii) the synergism existing between proteinases activity. We hypothesize that if one was to take these additional points in consideration when selecting candidate-compounds, it would be possible to use as few as a single proteinase inhibitor as efficient chemotherapy drug. In this work, we propose an in silico approach to identify inhibitors acting over a broad spectrum of Leishmania (V.) braziliensis proteinase classes, an important causative agent of mucocutaneous leishmaniasis in Brasil. We have searched the L. (V.) braziliensis cysteine-, metallo-, and serine proteinases (including all their isoforms) annotated in the databank GeneDB. To this end, representative sequences of proteinases of each class were aligned to sequences in the databank using the ALIGN EMBOSS server, to identify annotated sequences with high identity scores to the representative ones. The results identified 63 cysteine-, 96 metallo-, and 22 serineproteinases. Among cysteine proteinases we were able to identify 12 isoforms and, also, 13 in metallo proteinases, all with identity > 60%. Three dimensional (3D) models representatives for each isoforms group were built by homology and the modeled proteins structures were validated by Procheck programs: ERRAT and PROVE WHAT-CHECK. In the sequence, compounds with structure similar to inhibitors of all studied proteinase classes were retrieved from in ZINC database. Only compounds with a similarity rate of at least 60% to the inhibitors were selected. This strategy was able to retrieve 790 potential inhibitors of cysteine-, 9 of metallo- and 1357 of serine-proteinases. Additionally, the chemical structures of each group of inhibitors were aligned using Ligand.info databank, to identify the main chemistry structures of inhibitors of each proteinase class. The selected compounds were subjected to molecular docking and the
results indicate that some of them can have a multivalency inhibitory affinity over distinct groups of a proteinase class. Such observation does not apply to serine-proteinases, as enzymes of this class have a higher variability making it difficult for a single inhibitor to act on multiple enzymes of this proteinase class. Financial support: CNPq, CAPES and FAPERJ.
- CLINICAL LEISHMANIASIS [1016] P 551 - CLINICAL COMPARISON OF LOW AND REGULAR ANTIMONY DOSES THERAPIES LONG TIME AFTER TREATMENT OF CUTANEOUS LEISHMANIASIS IN RIO DE JANEIRO STATE, BRAZIL VIEIRA-GONCALVES, R.1; HERINGER, J.F.1; NOGUEIRA, R.S.1; GOMES-SILVA, A.1; DACRUZ, A.M.1; OLIVEIRA-NETO, M.P.2 1.LABORATÓRIO INTERDISCIPLINAR DE PESQUISAS MÉDICAS/IOC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.IPEC/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:leishmaniasis; antimony; therapy
Abstract: Introduction: Cutaneous leishmaniasis (CL) is the most frequent clinical form of leishmaniasis in Brazil, whereas mucosal disease (ML) is rare. Pentavalent antimony (Sb+) is used for treatment since 1912, and may cause heart and kidney adverse effects. In Rio de Janeiro State (RJ) Leishmania (Viannia) braziliensis is the specie associated with CL. The regular dose (RD) recommended by the Brazilian Health Ministry (10-15mg of Sb+/kg/day/21 days) is effective to cure CL patients from RJ. However, low Sb+ doses (LD) (5mg Sb+/kg/day/30 days) are also effective. If LD therapy increases relapses or evolution to mucosal leishmaniasis (ML) long time after therapy is not known. Our goal was to show if LD is as effective as RD even long time after therapy. Methods: 310 CL healed patients from Rio de Janeiro (IPEC/FIOCRUZ) and Paraty (SMS Paraty) municipalities previously treated with LD (n=175) and RD (n=135) were included. Thirty-nine field visits were done for patient’s clinical and laboratorial evaluation. Results: 52 CL-LD patients and 30 CL-RD patients were found. The groups were examined 1125 (LD) and 10-27 (RD) years after treatment. During active disease, all patients had typical CL lesions and positive Montenegro Skin Test. The age ranged from 5-76 years old in LD and from 5-69 years old in RD groups. After treatment, 48 LD patients (92%) cured and 4 patients (7.5%) needed a second treatment with RD. All patients treated with RD had cured. Two patients, one in each group, developed ML one year after treatment and were cured with 20mg Sb+/kg/day and amphotericin-B. The mean total Sb+ accumulated dose in the LD patients was 8,574 mg/patient. If they were treated with RD it would be 18,479 mg/patient. Conclusion: no drug resistance was observed in patients treated with RD after an initial LD. The cure was reached in 92% of cases with less than half of the recommended dose. Besides the easier administration, lower dose cause less adverse effects. A mean economy of $93.00/patient was achieved. One LD patient developed ML. Anyhow, this form occurs in 3-5% of all CL cases. We believe that CL resistance to Sb+ may occur independent of the used dose since RD therapy may occasionally fail. These results showed that in RJ patients, LD Sb+ therapy was as efficient as RD to clinically cure CL even long time after therapy (11-25 years), at least with L. braziliensis strains that circulate in Rio de Janeiro State. Support: IOC/FIOCRUZ, CNPq, FAPERJ. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1017] P 552 - PCR SCREENING OF VISCERAL LEISHMANIASIS BY CONJUNCTIVAL SWAB IN DOMICILED DOGS OF NORTH REGION OF BELO HORIZONTE, MINAS GERAIS STATE, BRAZIL. LEITE, R.S.; BARBOSA, A.D.; FERREIRA, A.L.C.; ANDRADE, A.S.R.
CENTRO DE DESENVOLVIMENTO DA TECNOLOGIA NUCLEAR, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; conjunctival swab; molecular diagnosis
Abstract: PCR assays have greatly improved the sensitivity of visceral leishmaniais (VL) diagnosis in dogs. Various canine tissues (including blood, urine, skin biopsies, lymph node, bone marrow and spleen) have been used for PCR detection of the parasite. However, the non invasive samples assume great importance in this context because they are simpler, painless and more easily allowed by the dog-owners. Non invasive samplings would represent an essential tool in mass-screening survey for interventional programs. An interesting approach in this context is the conjunctival swab (CS), a method for sample collection that uses a sterile swab for sampling the dog conjunctivas. This method was shown to be highly sensitive when used for diagnosis of symptomatic and asymptomatic dogs. The purpose of this study was to evaluate the molecular diagnosis of canine visceral leishmaniasis using conjunctival swab (CS) samples in dogs living in a highly endemic area and to compare the results with those obtained by the conventional serological tests used by the Brazilian VL control program. Eight hundred and seventy-seven dogs domiciled in the North Region of Belo Horizonte city (Minas Gerais state, Brazil) were screened. In parallel to the annual canine serological survey, in which blood samples were collected in filter paper, CS were also collected from both dogs conjunctives. The dogs were also clinically evaluated by a veterinarian. The DNA purification from CS was carried out by the phenol/chloroform method and after extraction DNA from both conjunctivas of the same dog were mixed constituting a single sample for the PCR assay. Leishmania infantum indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and real time PCR of CS samples were performed and the results were compared. The real time PCR was performed using Sybr Green and primers addressed to kDNA minicircles. Sixty-six percent (579/877) of dogs showed at least one symptom related to VL, being skin changes the main symptom. In the serologic assays 11% (97/877) of animals were ELISA positive and 5.2% (46/877) IFAT positive. In real time PCR assay 25% (222/877) were positive. The real time PCR using CS samples was highly sensitive and able to detect Leishmania infection in symptomatic or assymptomatic dogs with negative or positive serological diagnosis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1020] P 553 - QLOGP INFLUENCE IN ACTIVITY ANTILEISHMANIAL BY BENZOPHENONE DERIVATIVES ALVES, K.F.; DE ALMEIDA, L.; MACIEL-REZENDE, C.M.; PIRES, F.R.; DOS SANTOS, M.H.; MARQUES, M.J. UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL.
Keyword:leishmania; benzophenones; lipophilicity
Abstract: Leishmaniasis is a group of tropical diseases caused by species of protozoan parasites of the genus Leishmania. The drugs currently available for treatment of leishmaniasis are unsatisfactory because of their limited efficacy, frequent side effects, and increasing drug resistance. As a result of previous studies reveals that some natural benzophenones exhibit action on parasites such as Leishmania and Trypanosoma cruzi. The aiming of this work was correlate the leishmanicidal activity in promastigote forms of Leishmania (Leishmania) amazonensis with the calculated logP (QlogP) from benzophenone derivatives besides their starting compounds (CMA, CMB, CMC, LFQM-116 to LFQM-123). To evaluated the antileishmanial activity, benzophenones solubilized in DMSO were added to promastigotes of L. (L.) amazonensis (1x106 cells/ml ) in Schneider medium, performed in triplicate on three
separate occasions. Results were expressed as percentage inhibition compared to control. Lipophilicity values were estimated by determining the theoretical QlogP (octanol/water partition coefficient) using QikProp program. This software calculates the QlogP from regression equations using the experimental data and molecular descriptors (physical count of hydrogen bond, atom types and charges, etc.) through statistical mechanical simulations of Monte Carlo. The concentrations of 50% inhibition of growth (IC50) promastigotes with QlogP of starting benzophenones were CMA (29.00 µg/ml, 3.46); CMB (40.92 µg/ml, 3.07) and CMC (121.36 µg/ml, 2.71). The results presented by benzophenone derivatives were LFQM-115 (4.90 µg/ml, 3.79), LFQM-116 (9.80 µg/ml, 5.04), LFQM-117 (7.05 µg/ml, 5.29), LFQM-118 (5.05 µg/ml, 3.40), LFQM-119 (8.56 µg/ml, 4.60), LFQM-120 (7.82 µg/ml, 4.73), LFQM-121 (21.30 µg/ml,3.57), LFQM-122 (23.80 µg/ml, 5.71) and LFQM-123 (5.94 µg/ml, 6.21). From the results we can see that the QlogP of the compounds and the IC50 values show that the inclusion of groups resulted in derivatives with lipophilic profile more pronounced than their respectively precursors moreover, the benzophenone derivatives leishmanicidal activity on promastigotes forms, was similarly, most promising than the precursors. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1023] P 554 - HEMATOGENIC SPREAD OF LEISHMANIA (VIANNIA) IN NEUTROPHILS AND MONOCYTES ABREU, H.C.N.; CONTER, C.C.; PEDROSO, R.B.; SILVEIRA, T.G.V.; ARISTIDES, S.M.A. UNIVERSIDADE ESTADUAL DE MARINGÁ, MARINGÁ, PR, BRAZIL.
Keyword:leishmania; hematogenic spread; mononuclear cell
Abstract: After inoculation of Leishmania promastigotes in the host, the establishment of infection occurs involving phagocytosis and parasitism of neutrophils and macrophages. Since the persistence of Leishmania was already demonstrated, the possibility of finding Leishmania DNA in neutrophils and monocytes enables a better understanding of the pathogenesis of cutaneous leishmaniasis (CL). The objective was evaluating the presence of Leishmania (Viannia) DNA in polymorphonuclear and mononuclear blood cells from patients suspected of CL, using the Polymerase Chain Reaction (PCR). In order to achieve the cells, 6 mL blood with EDTA were collected and the reagent Mono-Poly Resolving Medium (MP Biomedicals, LLC) was used as prescribed by the manufacturer. After polymorphonuclear and mononuclear separation and washing, DNA was extracted by guanidine isothiocyanate and phenol. Were used MP3H and MP1L primers for amplification. Were studied 94 patients, 63.83% were men and, 36.17% women. The majority of patients (86-91.49%) had cutaneous lesions, with a predominance of single lesion (58.51%), and 8 (8.51%) presented mucosal lesion. From 94 patients, 71 (75.53%) had primary lesion and no history of CL and, 23 (24.47%) reported previous infection. From 71 patients with primary lesions and no history of LT, 30 (31.91%) were diagnosed with CL, according to the Health Ministry (Brazil). From 23 patients who reported previous infection, 7 (30.43%) presented reactivation of the primary lesion, 2 (8.69%) presented new skin lesion and, 14 (60.86%) returned for treatment control. PCR was positive for two patients who presented active primary lesion with 3 to 6 months of time evolution; these patients did not present history of CL and were diagnosed according to tests recommended by the Health Ministry (Brazil). The presence of Leishmania (Viannia) DNA in mononuclear and polymorphonuclear cells demonstrates the parasite persistence, considering the lesion time in both patients. This persistence indicates that, in some patients, the parasite is able to escape to the macrophage elimination mechanisms and remain in the body and, hematogenic spread can occur with development of mucosal lesion.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1026] P 555 - A NOVEL FLUORESCENT PROBE FOR THE STUDY OF LEISHMANIA SMALLMOLECULE PHARMACOLOGY BATISTA, A.J.S.1; OLIVEIRA, S.D.1; DUVAL, R.2; COUTINHO-SILVA, R.1; ROSSIBERGMANN, B.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UNIVERSITÉ TOULOUSE III, TOULOSE, FRANCE.
Keyword:leishmaniasis; fluorescent probe ; pharmacology
Abstract: We have devised a novel fluorescent probe derived from antiparasitary natural products, in order to study the small-molecule pharmacology of Leishmania in vitro. The new probe penetrates rapidly and with elevated specificity in L. amazonensis promastigotes, and allows stable fluorescent labeling of the treated cells. This probe thus demonstrates optimal features for the biological screening of natural and synthetic small molecules by fluorescence microscopy. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1027] P 556 - TOPICAL TREATMENT USING A CHLOROALUMINUM PHTHALOCYANINE (CLALPC)-NANOEMULSION AGAINST LEISHMANIA (VIANNIA) BRAZILIENSIS INFECTED BALB/C MICE ESCOBAR, P.1; LEAL, S.M.1; CARREÑO, H.1; JUNIOR, E.R.2; CORREA, J.R.3; DE AZEVEDO, R.B.3 1.UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA; 2.UNIVERSIDAD FEDERAL DE RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 3.UNIVERSIDAD DE BRASILIA, BRASILIA, DF, BRAZIL.
Keyword:topical treatment; chloroaluminum phthalocyanine; nanothechnology
Abstract: Nanoemulsions(NE) as delivery system is an interesting option for cutaneous leishmaniasis (CL) ensure better solubility and biodistribution of hydrophobic compounds. We determinate the efficacy of photodynamic therapy (PDT) using ClAlPc-NE topically in BALB/c mice infected with L(V).braziliensis. ClALPc-NE was prepared by ultrasonication and size, polydispersity index (PDI) and Zpotential were determined. Female BALB/c mice 8-10 weeksold were intradermically infected at the base of the tail with 2x106 L.(V)braziliensis stationary promastigotes. Forty five days after, animals were randomly separated. In Exp1 (two groups, 3 mice each), animals were treated with 100 µM of ClPcAl-NE/day at interval of 2 days, 4 times. Control mice were leaving without treatment. Fifteen minutes after each dose mice were anesthetized and irradiated for 20 min using a red lightemitting diode (LED). In Exp2 (5 groups, 4 mice each) mice were treated as followed: Group1, 200 mg/kg/day Glucantime/20 days intramuscularly; group2, 100 µM ClPcAl-NE/day, 10 times, two days interval; group3, 300 µM ClAlPc in DMSO:Tween 80:type I water, 5 times, two days interval; group4, NE without ClAlPc; and group5, without treatment. Three hour after each treatment, mice from 2-4 groups were irradiated as above. Animals were sacrificed 20 days after last dose. Lesion sizes were estimated weekly using a dial caliper and parasitological efficacy was determined microscopically. The ClAlPc-NEs form Exp 1 and 2 showed: size 129nm, PDI 0.1, Zpotential -13.3mV and size 311.4nm, PDI 0.403, Zpotential -23.5mV respectively. In Exp1, the lesion size before treatment was 22.51±11.08mm2 and at the end of experiment was 18.81±7.92 mm2 and 19.97±19.25 mm2(control). In Exp2 lesion areas before and at the end of treatment were: Grup1, 31.97±8.92mm2 and 107.61±7.80mm2 (nodule)/44.17±4.80mm2 (ulcer); group2, 62.06±8.16mm2 and 211.74±23.14mm2 (nodule)/107.90±16.02mm2 (ulcer); grup3,
20.25±6.57 mm2 and a necrosis in tail and leg at the end. Control groups (4 and 5) lesion areas were 161.23±53.65mm2 (nodule)/63.80±17.74mm2(ulcer) at the end. In all stained slides abundant intracellular and extracellular parasites were observed in skin preparation but not in liver or spleen. The lack of ClAlPc-NE efficacy reaffirm the difficulties for development of CLtopical treatment by L.(V)braziliensis. More dosages are recommended however a precaution in order to avoid necrosis or deleterious reactions is recommended.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1028] P 557 - STRUCTURAL AND FUNCTIONAL STUDIES OF LEISHMANIA MAJOR NUCLEOSIDE DIPHOSPHATE KINASE DE OLIVEIRA, A.H.C.1; DIAS, F.C.2; WARD, R.J.1; PINTO, M.R.1; VIEIRA, P.S.1 1.FFCLRP-USP, RIBEIRÃO PRETO, SP, BRAZIL; 2.FMRP-USP, RIBEIRÃO PRETO, SP, BRAZIL.
Keyword:enzyme; mutagenesis; overexpression
Abstract: The nucleoside diphosphate kinase (EC 2.7.4.6; NDK) is ubiquitous in both prokaryotes and eukaryotes catalyzing the transfer of the gama-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate by a ping-pong mechanism. Besides the involvement of this enzyme in maintaining the nucleotides cellular pool, it was described in other cellular processes, and is considered a multifunctional enzyme. Studies have demonstrated that NDKb is secreted by intracellular pathogens during infection, including by Leishmania, preventing ATP-induced cytolysis of macrophages, thereby preserving the integrity of host cells to the benefit of the parasite. Our group has previously shown NDKb as an abundant component of the microsomal fraction of L. major promastigotes by subproteomic analysis (de Oliveira et al., Comp. Biochem. Physiol. Part D, vol. 3, 2006). Thus, the characterization of L. major NDK (LmNDK) may contribute to understand the functions of this protein in parasite/host interactions. The LmNDK coding sequence was amplified genomic DNA, cloned into pET28a and modified by sitedirected mutagenesis. The recombinant LmNDK and mutants R17A, E28A, P95S, P100S-Δ5 and Δ5C-term were efficiently expressed as soluble form in E. coli and purified by affinity chromatography. The solution characterization was carried out with the recombinant LmNDK and the P95S, P100S-Δ5 and Δ5C-term mutants. Circular dichroism and intrinsic fluorescence of tryptophan emission experiments showed LmNDK is more stable than P95S and P100S-Δ5 mutants. The catalytic activity analysis showed the P95S had a similar LmNDK activity, but other mutants (R17A, E28A, P100S-Δ5 and Δ5-Cterm) exhibited a decreased activity. All proteins presented a cytotoxic effect in the ATP presence, but this effect was reduced in the acceptor presence. Polyclonal antibodies raised against the native protein from the promastigote extracts and the subcellular localization, using fluorescence microscopy, demonstrated that the native NDK was localized in the promastigote cytoplasm. L. major (LV39) transfectant overexpressing LmNDK presented partial virulence attenuation in the BALB/c footpad lesion. Preliminary comparative analysis of 2D-gels protein profile of transfectants revealed differences of promastigote protein expression, including the NDK overexpression. Supported by FAPESP, CNPq, PRP-USP.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1031] P 558 - ENCAPSULATION IN LIPID CORE NANOCAPSULES INCREASES THE ORAL EFFICACY OF QUERCETIN AGAINST CUTANEOUS LEISHMANIASIS
SOUSA-BATISTA, A.J.1; POLETTO, F.2; POHLMANN, A.R.2; GUTERRES, S.S.2; ROSSIBERGMANN, B.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRGS, PORTO ALEGRE, RS, BRAZIL.
Keyword:nanotecnology; leishmaniasis ; chemoterapy
Abstract: Our group has previously described the in vivo activity of quercetin (QC) against Leishmania amazonensis when administered by the oral route (Muzitano, M.F., et. al., 2008). With the purpose to improve antileishmanial efficacy we encapsulated QC and its pentacetylated derivative (PQC) in lipid core poly(epsilon-caprolactone) nanocapsules (LNC), prepared by interfacial deposition of preformed polymer. To test those formulations, BALB/c mice were infected in the ear with 2x106 L. amazonensis GFP-promastigotes. After 7 days of infection the oral treatment was initiated daily for 52 days with either the free drugs (QC and PQC) or their LNC formulations (LNC-QC e LNC-PQC, respectively). The ear lesion sizes were measured throughout the infection with a dial caliper. On day 59 of infection, the animals were sacrificed and the parasite load in the lesions was quantified both by Limiting Dilution Assay and by fluorimetry. We observed that nanoencapsulation increased drug effectiveness by 40-fold in relation to free drugs. To evaluate if the formulations induced cardiac, liver or kidney toxicity, the concentrations of aspartate aminotransferase (TGO), alanine aminotransferase (TGP) and creatinine were measured in the serum. None of the formulations tested showed detectable toxicity. These results show that the nanocapsules raised the efficacy of free QC and PQC in the treatment of cutaneous leishmaniasis through a non-invasive oral administration. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1032] P 559 - THE ANTILEISHMANIAL EFFECT AND SKIN ABSORPTION STUDIES OF LIPID-CORE POLY- €-CAPROLACTONE NANOCAPSULES ENTRAPPING THE CHALCONE CH8 USING BIOLUMINESCENT MICROSCOPY. LOPES, M.V.1; POLETTO, F.2; FERRARINI, S.R.2; MEDEI, E.H.1; GUERRA, B.1; GUTERRES, S.S.2; POHLMANN, A.R.2; GOMES, A.M.O.1; BERGMANN, B.R.1 1.UFRJ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRGS, PORTO ALEGRE, RS, BRAZIL.
Keyword:tegumentar leishmaniasis; topical treatment; nanocapsules
Abstract: No effective topical treatment exists for cutaneous leishmaniasis in many parts of the world. This is mainly due to the difficulty of an active drug to cross the tickened skin barrier. Over the last years, new drug delivery systems have been investigated to promote increased skin permeation. In this study we evaluated the efficacy of lipid-core poly-€-caprolactone nanocapsules entrapping the antileishmanial chalcone CH8 (LNC-CH8) to treat cutaneous leishmaniasis by the subcutaneous (sc) route. Also, by using fluorescent rhodamine-labeled LNC-CH8 (LNCF-CH8), we conducted bioluminescence studies to evaluate macrophage internalization and skin absorption across the skin. The LNC-CH8 and LNCF-CH8 nanoformulations measuring 210 nm were prepared by interfacial deposition of rhodaminecoupled preformed polymer. The antileishmanial activity of LNC-CH8 was first evaluated in vitro against L. amazonensis promastigotes cultured with varying concentrations of LNC-CH8, free CH8 or empty LNC. The number of parasites was counted after 48h of culture at 27oC. The results showed that like free CH8, LNC-CH8 inhibited promastigote growth at IC50 = 1.2 µg/mL. For in vivo efficacy, BALB/c mice were infected in the ear and from day 7 were treated twice a week for 3 weeks with 5 µg of CH8 in LNC or in its free form. LNC-CH8 s.c. treatment effectively controlled parasite growth in the infected ears as compared to untreated and free CH8-treated mice. To evaluate macrophage uptake, non-infected and L. amazonensis-GFPinfected macrophages were incubated for 2 hours with LNCF-CH8. Images of red-fluorescent
particles and green-fluorescent parasites were taken by intravital multiphoton microscopy, showing LNCF-CH8 co-localized with the parasites inside macrophage vacuoles. To evaluate skin absorption, LNCF-CH8 was topically applied onto shaved dorsum of mice. At different times after application, bioluminescent images were taken with IVIS LUMINA, showing a decreased fluorescence indicative of permeation at 120 min of LNCF-CH8 topical application. In all, these results demonstrated that lipid-core LNC-CH8 nanocapsules are effective against L. amazonensis in vitro and in vivo, can deliver the lipophilic drug CH8 into the parasitophorous vacuoles of macrophages, and can permeate mouse skin, indicating potential use in topical treatment of cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1033] P 560 - EX VIVO PERMEATION AND RETENTION OF CHOLOALUMUNINUM PHTHALOCYANINE (CLALPC) NANOEMULSION IN HEALTHY HUMAN SKIN LAYERS, FOR OPTIMIZATION OF TOPICAL FORMULATIONS FOR CUTANEOUS LEISHMANIASIS ESCOBAR, P.; OSPINA, V.E.; CONDE, C.A.; MANTILLA, J.C. UNIVERSIDAD INDUSTRIAL DE SANTANDER, BUCARAMANGA, COLOMBIA.
Keyword:franz diffusion cells; topical treatment; nanothechnology
Abstract: The ClAlPc, a phototosensitizer used in photodynamic therapy (PDT), constitutes an important alternative for cutaneous leishmaniasis (CL) treatment. The ClAlPc topically applied must penetrate skin layers and be retained in dermis, place where Leishmania-infected macrophages reside. The aim of this study was to determine the permeation and retention of ClAlPc, incorporated in a nanoemulsion (NE) delivery system, in healthy human skin membranes .The ClAlPc-NE (oil-to-water) was prepared by ultrasonication techniques. The average size was 195.2 nm, polydispersity index 0.235, zeta potential -18.8mV, ClAlPc concentration 256.96 µM and phospholipids concentration 4788 nmol/mL. Abdominal skin was obtained from healthy women under gone cosmetic surgery (Ethics Committee approval). The skin was washed, cleaned for subcutaneous fatty tissue and storage at -20°C up to 2 months. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to ClAlPc-NE for 0, 4, 8, 10 12 and 24 h. PBS 7.4 SDS 2% was used as receptor fluid. Two different skin donors were used. Six assays (6 cells each) were made. Skin integrity before and after experiments were analyzed from skin biopsies stained with hematoxylin-eosin. ClAlPc retained in stratum corneum (SC) was analyzed by skin surface stripping with adhesive tape (15 tapes). Each tape was placed for 30 seconds on the membrane and transferred to ClAlPc extraction-solvent for 24 h. ClAlPc retained in epidermis and dermis (E+D) was analyzed from a macerate of the remaining membrane tissue transferred for extraction as above. The ClPcAl concentration was analyzed by spectrofluorimetry and results were expressed as nM/cm2. The ex vivo treatment of human membranes with ClAlPc-NE in Franz diffusion cells was unable to detect, at any point of time, ClAlPc concentration in the receptor medium indicating that ClAlPc could not completely through the skin. The ClAlPc was retained mainly in SC with average values of 42.03 nM, and in E+D with values of 7.3 nM. Histological changes were noted in the skin after testing such as: SC detachment, collagen fragmentation and changes in the conformation of the epidermis stratified epithelium. The results showed that although ClAlPc contained in a NE could not diffuse across the skin, it could penetrate and be retained in the different parts of the skin (SC, E, D). The greater retention SC compared with E+D sugested than NE should be improved to provide a greater flow of the compound into deeper layers of the skin, promoting greater ClAlPc retention in the dermis.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1041] P 561 - ANTI-LEISHMANICIDAL ACTIVITY OF THE ANTIMICROBIAL PEPTIDE DRS01 OBSERVED IN LEISHMANIA INFANTUM (SYN. LEISHMANIA CHAGASI) CELLS BY ATOMIC FORCE MICROSCOPY SILVA, V.C.1; EATON, P.2; BITTENCOURT, C.R.3; VERAS, L.M.C.3; COSTA, C.H.N.1; FEIO, M.J.F.2; DE ALMEIDA LEITE, J.R.S.3
1.LABORATÓRIO DE PESQUISAS EM LEISHMANIOSES DO INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELLA, TERESINA, PI, BRAZIL; 2.REQUIMTE, DQB, FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DO PORTO, PORTO, PORTUGAL; 3.NÚCLEO DE BIODIVERSIDADE E BIOTECNOLOGIA DA UNIVERSIDADE FEDERAL DO PIAUÍ, PARNAÍBA, PI, BRAZIL.
Keyword:antimicrobial peptide; leishmania infantum; atomic force microscopy
Abstract: Background: Leishmaniasis is one of the most serious diseases in the world, with inadequate medication, especially for visceral leishmaniasis, commonly caused by Leishmania (L.) infantum. A recently described antimicrobial peptide DRS 01 has been reported to kill L. infantum promastigotes, but nothing is known about its mode of action or effect on the cell. Methods: The Dermaseptin 01 (DS01) isolated and identified from frog skin secretions of Phyllomedusa hypochondralis was synthesized in an automated peptide synthesizer (PSSM 8, Shimadzu) by solid-phase synthesis according to the F-moc procedure and purified by RPHPLC. Leishmania infantum live cells were treated for 24 h with 16, 64 and 128 µg/mL of DRS 01, concentrations chosen to be below, around and above LC50, and samples were prepared for microscopic observation. Results: In the AFM images of the treated cells, considerable differences were found compared to the control. In general disturbances were found on the membranes of the cell body, and often the cells appeared less elongated after treatment. The surface roughness increased with treatment with the antimicrobial peptide. The roughness, as measured by Rq, almost doubled from 3.4 ± 0.8 nm for the control cells to 7.1 ± 2.1 nm for the cells treated with 128 µg/mL DRS 01. Conclusions: For the first time, high-resolution microscopy has been carried out on promastigote forms of Leishmania infantum, and revealed important morphological details of the parasite. The results suggest than the mode of action of DRS 01 is directly on the membrane, presumably leading to cell lysis. The implication of a membrane-directed attack is that resistance will be difficult for the parasite to develop, and that the specificity towards the target cells is likely to be high. Supported by FCT/CNPq Bilateral project Brasil/Portugal, FCT project PEst-C/EQB/LA0006/2011 and Nanobiomed Network CAPES/Brasil.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1043] P 562 - GOLD NANOROD-BASED LOCALIZED SURFACE PLASMON RESONANCE BIOSENSOR FOR SENSITIVE DETECTION OF ANTIBODY ANTI-LEISHMANIA INFANTUM IN CANINE SERUM SESANA, A.M.; DE JESUS, A.C.; ANDRADE, H.M.; LADEIRA, L.O.; GAZZINELLI, R.T. UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:nanotechnology; visceral leishmaniasis; diagnosis
Abstract: Up to date, immunoassay plays a prominent role in the early diagnosis of infectious disease. As the current gold standard, enzyme-linked immunosorbent assay (ELISA) has been one of the
most extended methods for the serological diagnosis in immunogenic protein detection. It still has some shortcomings, such as long testing time needed, indirect format of detection and multiple washing steps. Efforts have been made to increase their detection limits, however, progress has been disappointing especially in quantitative analysis. Recently, there have been extensive efforts to develop nanoparticles-enhanced analytical technologies for the highly sensitive and selective detection of protein biomarkers that can be powerfully utilized to diagnose various types of diseases. Gold nanorods (GNRs) functionalized with antibodies can also be used in sandwich assays to greatly enhance the sensitivity of measurements for the ultrasensitive detection of protein biomarkers, based on the surface plasmon resonance (SPR). Functionalization of the GNRs with antibodies or other biomolecules allows their specific attachment to any target cell. This is a useful attribute for biomedical diagnostic applications. In this study, it has been taken as the example for the antibody detection, a hypothetical protein of Leishmania infantum labelled on the surface of the GNRs through covalent adsorption. A hypothetical protein has previously chosen interesting results in terms of sensitivity and specificity in ELISA tests for the diagnosis of disease in canine serum. Some characterizations have been conducted to access the nature of the GNRs after biological modification. In addition, a validation experiment also has been designed in a 96-well microplate based on ELISA to evaluate the influence of steric effects on the binding affinity of the antibody–antigen pair. The use of the hypothetical protein of Leishmania infantum associated with the gold nanorods enable up to a further 102 times increase in sensitivity of the diagnosis test compared with the ELISA method. The technique presented in this study can play a key role in developing tunable sensors for sensitive and precise monitoring of different biological interactions.
- BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1044] P 563 - ANTILEISHMANIAL ACTIVITY OF NEW QUINONE DERIVATIVES OF LAPACHOL REMPEL, S.S.1; GOMES, S.2; CANTO-CAVALHEIRO, M.M.1; DA SILVA, A.J.M.2; COSTA, P.R.R.2; CUNHA-JÚNIOR, E.F.1; CAMPOS-SANTOS, E.C.1 1.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL; 2.UFRJ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:chemotherapy; leishmania amazonensis; quinone
Abstract: Considered by World Health Organization (WHO) a neglected disease, leishmaniasis affects over 12 million people in the world per year. Treatment receives low investment from the pharmaceutical industries, requires long periods of administration and presents low efficacy, resistance, toxicity and high cost. Quinones represent a very important class of molecules which is directly related to many metabolic processes in the cell and presents trypanosomicidal effect. The purpose of this study is to evaluate the activity of 12 derivatives from lapachol (LQBs) in promastigotes and intracellular amastigotes of L. amazonensis and their toxicity in murine macrophages. Promastigotes (1x106/ml) were incubated with different concentrations of LQBs or none by 72 h and the parasite viability was analyzed by MTT assay. The activity on intracellular amastigotes was evaluated by light microscopy in L. amazonensis-infected murine macrophages after incubation with several concentrations of LQBs for 72 h. Toxicity was evaluated in murine macrophages by MTT assay after 72 h of incubation. The differences on the structure of each substance were related to some aspects of the drug action to the cells. The closing of the ring in "3" rendered low toxicity and antiamastigote activities, while the addition of a diethylformamide in "2" gives higher activity to intracellular amastigotes but also higher toxicity. On the other hand, when both modifications took place in the same compound, a series with selective antiamastigote activity was originated, from which LQB-270 presented the lowest IC50 (1.5 µM) on intracellular amastigotes and selectivity index high. This structure-activity
study pointed LQB-270 as a promising candidate to be evaluated in experimental cutaneous leishmaniasis. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1051] P 564 - NEW THIOSEMICARBAZONES DERIVATIVES: IN VITRO EFFECTS ON LEISHMANIA AMAZONENSIS AND IMMUNE SYSTEM CELLS SANTOS, T.A.R.1; SILVA, A.C.1; SILVA, E.B.2; MOREIRA, D.R.M.2; LEITE, A.C.L.2; PEREIRA, V.R.A.1 1.CENTRO DE PESQUISAS AGGEU MAGALHÃES, RECIFE, PE, BRAZIL; 2.UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE, PE, BRAZIL.
Keyword:thiosemicarbazones; leishmania amazonensis; immune system
Abstract: Thiosemicarbazones derived molecules are known for its remarkable biological effects, with studies showing its antitumoral, antibacterial and antiprotozoal activities. Several drug screenings with tripanossomatidae family members have been conducted, demonstrating these molecules to have special properties against parasites, particularly in Trypanossoma cruzi. The aim of this work was to assess the impact of five new thiosemicarbazone derived molecules (coded AT1, AT2, AT3, AT4 and AT6) on immune system cells, and its effects on Leishmania amazonensis. For this purpose, BALB/c splenocytes were collected and incubated in 96 wellplate with different molecules concentrations (1 to 100 µg/mL) and 1 µCi of tritiated thymidine was add to each well. After 18 hours, cells were harvested and the incorporation of triatiated thymidine was measured with a beta counter. Cell viability after exposure to the compounds was evaluated in J774A.1 macrophages by MTT assay. The 50% cytotoxicity concentration (CC50) values were determined to splenocytes and macrophages. To analyze the direct effect of compounds in L. amazonensis, promastigotes (106 parasites/mL) were incubated with molecules in different concentrations (0.19 to 100 µg/mL) and after 96 hours cells were counted in Neubauer chamber and the concentration that inhibited culture growth by 50% (IC50) was calculated. Selectivity Index (SI) were determined by the ratio of CC50 (Macrophages) to IC50 (Promastigotes). Among the analyzed compounds, AT2 and AT6 showed the lowest IC50 values (1.40 and 1.99 µg/mL respectively) and the highest CC50 values either in splenocytes (18.28 and 14.49 µg/mL) or macrophages (12.73 and 12.97 µg/mL). Consequently these two molecules presented the highest SI values (9.09 and 6.52 respectively) demonstrating more selectivity to the parasites than the mammalian cell. AT1, AT3 and AT4 also showed antiparasite activity, with IC50 values varying from 1.49 to 3.11 µg/mL and SI values from 2.29 to 4.54. The leishmanial activity in relatively low level and fewer effects on immune system cells endorse this series of molecules as promising candidates to drugs against leishmaniasis, stimulating new studies. - BIOCHEMISTRY, CHEMOTHERAPY, DRUG DEVELOPMENT AND DRUG-RESISTANCE [1053] P 565 - LEISHMANIA (L.) AMAZONENSIS PROMASTIGOTE MUTANT SELECTED IN VITRO FOR RESISTANCE TO SB(III) CONFERS RESISTANCE TO PENTAVALENT ANTIMONIAL THERAPY IN VIVO. REIS, P.G.1; MONTE NETO, R.L.D.2; BEZERRA, F.M.B.V.1; NEVES, F.T.P.1; MELO, M.N.3; FRÉZARD, F.1
1.DEPARTAMENTO DE FISIOLOGIA E BIOFÍSICA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.CENTRE DE RECHERCHE EN INFECTOLOGIE, UNIVERSITÉ LAVAL, QUÉBEC-, CANADA; 3.DEPARTAMENTO DE PARASITOLOGIA, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL.
Keyword:l. (l.) amazonensis; antimony resistance ; in vivo
Abstract:
Leishmania promastigote mutants selected in vitro for resistance to Sb(III) have been used extensively as a model of investigation of the mechanisms of resistance in antimonial chemotherapy. However, this model is often questioned with respect to its relevance to in vivo condition, as it is based on the assumption that Sb(III) is the final active form in antimonial chemotherapy and that resistance to pentavalent antimonials results from resistance of Leishmania parasite to Sb(III). Support to this hypothesis was obtained from in vitro assays using the macrophage model, showing cross-resistance to Sb(V) of intracellular Leishmania strains initially selected for resistance to Sb(III). However, the macrophage model is still far from the in vivo condition and it would be important to investigate in vivo whether Leishmania promastigote mutants selected in vitro for resistance to Sb(III) confer resistance to pentavalent antimonial therapy. As a first step, the infectivity of Sb(III)-susceptible (WTS) and SbIIIresistant (SbR) populations of L. (L.) amazonensis was determined by inoculation of 1 × 106 metacyclic promastigotes from the sixth day of the stationary phase into the footpads of Balb/c mice. After appearance of the lesion, groups of six animals infected by each strain were treated with meglumine antimoniate for 30 days and other groups remained without treatment. The lesion growth was measured, and after the treatment, the animals were sacrificed and parasite load was determined using a quantitative limiting-dilution assay. The rates of increase of footpad lesion were similar in WTS and SbR groups, demonstrating the maintenance of infectivity of the resistant strain after in vitro selection. Pentavalent antimonial treatment promoted the stabilization of lesion growth in WTS group (3.8 mm) as opposed to SbR group, whose lesions evolved similarly to untreated SbR group (6 to 8mm). The in vivo resistance was also confirmed by parasite loads in the lesion, with SbR group showing 1028 and 1028 parasites with or without treatment, respectively (WTS showed 104 and 1012 with and without treatment). In conclusion, L. (L.) amazonensis promastigote mutant selected in vitro for resistance to Sb(III) confers resistance to pentavalent antimonial therapy in vivo. This in vivo model should be useful in the search of reversion strategies for antimonial resistance in L. (L.) amazonensis. Supported by : CNPq; Capes; Fapemig
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1059] P 566 - FLOW CYTOMETRY ON THE DIAGNOSIS OF PATIENTS WITH AMERICAN TEGUMENTARY LEISHMANIASIS (ATL) THROUGH RESEARCH ON IGG ANTIBODIES OF L. (V.) BRAZILIENSIS DE OLIVEIRA, A.P.; DE OLIVEIRA, B.C.; CASTRO, M.C.A.B.; DE ALMEIDA, A.F.; SOUZA, M.A.; DE ALMEIDA, T.M.; SILVA, A.C.; DE BRITO, M.E.F.; PEREIRA, V.R.A. CPQAM-FIOCRUZ, RECIFE, PE, BRAZIL.
Keyword:atl; igg; flow cytometry
Abstract: ATL is a serious public health issue in Brazil. Besides the socioeconomic challenges, it is known that the disease’s diagnosis does not present a laboratorial method which can be used as gold standard. Until now, ATL’s diagnosis is based on clinic, epidemiologic and laboratorial factors, so the association of several elements is frequently necessary to reach the definitive diagnosis. Therefore, the development of new diagnostic methods is essential, and flow cytometry is a new approach which is utilized on the research on antibodies with superior applicability than different conventional protocols of detection and development. Taking flow cytometry’s applicability into consideration, this study brought an alternative through the research on IgG antibodies of L.(V.)braziliensis, which is applicable to ATL’s diagnosis. Serum from 20 patients before treatment (BT) were diluted (1:64 to 1:8192), incubated with L.(V.)braziliensis parasites and marked with human anti-IgG conjugated with fluorescein
isothiocyanate - FITC.The preview of the results was accomplished on the flow cytometer FACScalibur using the software CELL QuestTM, having the results expressed on levels of IgG’s reactivity, through the percentage of positive fluorescent parasites (PPFP).They were analyzed using the statistics program MedCalc. Using 20% of PPFP, cut-off suggested by Martins-filho et al, 1995, the results pointed that at the 1:128 cut-off, it is the reactivity region which corresponds to the first titer of differential reaction from BT individuals. Thus, it was shown 80% (16/20) of BT individuals located on a high reactivity region, in other words, positive for ATL; and 20% (4/20) of after treatment (AT) individuals with restricted PPFP values from a low reactivity region, meaning negative for the flow cytometry test. However, flow cytometry proved to be an applicable methodology to ATL’s diagnosis, suggesting a greater explanation of the disease, identifying cases of active ATL. In addition, further studies will elucidate the role of IgG antibodies on the hummoral immune response on ATL’s diagnosis and cure criterion. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1060] P 567 - COMPARISON OF THREE SEROLOGICAL TESTS FOR THE DIAGNOSIS OF THE CANINE VISCERAL LEISHMANIASIS IN THE CITY OF PORTEIRINHA, AN ENDEMIC AREA IN THE STATE OF MINAS GERAIS, BRAZIL. FRANÇA-SILVA, J.C.1; HORACIO, A.M.L.2; ROCHA, A.M.S.2; REIS, V.W.2; REGINA-SILVA, S.3; MICHALSKY, E.M.3; DIAS, E.S.3; MACHADO-COELHO, G.L.2 1.UFMG, BELO HORIZONTE, MG, BRAZIL; 2.UFOP, OURO PRETO, MG, BRAZIL; 3.CPQRR/FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; kala-azar detect; porteirinha
Abstract: The canine visceral leishmaniasis (CVL) is a serious public health and veterinarian problem in Brazil. The disease control is extremely complex and with considerable cost. Canine diagnosis and euthanasia are very discussed in terms of effective disease control. The present study aim to determine the incidence of CVL in the city of Porteirinha (Minas Gerais state) and started after a first canine census survey performed in 2008. The total number of dogs included in that study was 1,108. Canine serum collection was performed along time at 73, 162, 252, 342, 437 and 497 days and those sera were assayed by three different methods for VL diagnosis: indirect immunofluorescence (IFA, with 1:40 dilution cut off), ELISA (expressed by A450nm) and kalaazar detect rapid (KD-rK39) assays. According to the results, five groups of dogs were defined: 1. Confirmed (all three tests positive); 2. Undetermined (only ELISA positive); 3. Negative (all three tests negative); 4. Suspected (ELISA and IFA or KD positive); 5. Gray zone (A450nm ≤20% of the ELISA cutoff). The positivity rates were expressed as percentages and the incidence rates as new cases per 1,000 dogs per year. In group 1, the highest incidence was 3.5 at 342 days and the lowest one was 1.0 at 437 days. For group 2, the highest incidence was 18.9 after 252 days and the lowest one was 6.4 at both 162 and 437 days. The highest incidence observed in group 4 was 6.2 after 252 and 497 days and the lowest one (3.0) occurred at the 437th days. For the gray zone, the highest incidence was 4.4 at 342 days and the lowest one was 1.1 after 497 days. In group 3, the positivity rates displayed a uniform tendency to decrease, starting with 84% (73 days) and ending with 39.9% (497 days). The sensitivity of the KD test was 48% and the specificity was 81% (p0.0001). Although the KD test is extremely useful in the field, in an overall analysis with all the times together it displayed a low sensitivity (31%). This data indicates that the test needs improvements for better performance.
Financial support: FAPEMIG, FIOCRUZ, UFMG, HERTAPE-CALIER, CNPq. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [1065] P 568 - DIAGNOSTIC OF VISCERAL CANINE LEISHMANIASIS BY SEROLOGICAL METHODS IN NON-ENDEMIC REGION OF MINAS GERAIS. JUNIOR, J.G.C.1; FREIRE, M.L.1; CAMPOS, S.P.1; CUPOLILLO, E.2; DE ALMEIDA, R.P.2; SCOPEL, K.K.G.1; COIMBRA, E.S.1 1.UFJF, JUIZ DE FORA, MG, BRAZIL; 2.FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral canine leishmaniasis; diagnosis ; serology
Abstract: Introduction: Human visceral leishmaniasis constitutes a public health problem that affects millions of people throughout the world. In Brazil, visceral leishmaniasis (VL) is an endemic zoonotic disease caused by L. infantum. In general, VL is a rural disease, but nowadays this disease has spread to the urban centers. Dogs play an important role in the maintenance of this disease in the human environment serving as reservoirs for this infection. This study aimed to carry out a serological survey on canine visceral leishmaniasis (CVL) at Juiz de Fora, Minas Gerais. Methods: Blood was collected from 401 dogs allocate in the public kennel from municipality of Juiz de Fora. Diagnosis of CVL was performed using the colloidal gold-based immunochromatography TR DPP® kit, and EIE-leishmaniose-visceral-canina® kit, both produced by FIOCRUZ (Bio-Manguinhos, RJ, Brazil). All the procedures were performed according to the manufacturer’s instructions. In parallel, questionnaire with animal clinical aspects was filled out by a veterinarian. Dogs were scored for asymptomatic, oligosymptomatic and symptomatic. Results and Conclusion: From 401 dogs examined, 3.74% showed positive by TR DPP® kit, and 1.74% were positive by EIE-LVC. However, only 0.25% were positive for both diagnostics methods. Among the positive dogs only 1% was classified as oligosymptomatic. This is a first study about CVL, caused by L. infantum, in Juiz de Fora municipality and the results reinforce the idea that this disease presents a process of expansion and urbanization in Brazil. Furthermore, DPP technology represents a breakthrough in the detection of positive dogs from indene areas of CVL. These results show the importance of diagnosing CVL in non-endemic area and reinforce the necessity of epidemiological survey in Juiz de Fora. Supported by FAPEMIG, CNPq and UFJF. - CLINICAL AND EXPERIMENTAL IMMUNOLOGY [1073] P 569 - LEVELS OF ANTI-LEISHMANIA IGG3 CAN BE ASSOCIATED WITH THERAPEUTIC PROGNOSTIC OF VISCERAL LEISHMANIASIS IN CHILDREN. OLIVEIRA NASCIMENTO, J.M.1; SILVA, G.A.F.2; DORVAL, M.E.C.1; OSHIRO, E.T.1; FRAGA, T.L.1; BRUSTOLONI, Y.M.1; SILVA, A.G.2; DA-CRUZ, A.M.2; DE OLIVEIRA, A.L.L.1 1.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL, CIDADE UNIVERSITÁRIA, 79070-900, CAMPO GRANDE, MS, BRAZIL; 2.FUNDAÇÃO INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral leishmaniasis; antibodies; humoral response
Abstract: The clinical expression of visceral leishmaniasis (VL) depend on factors such as species, parasite strain's virulence, individual, genetic and environmental characteristics and immunologic system responsiveness. The presence of antibodies reactive to Leishmania chagasi antigen by polyclonal activation of B cells is reported in literature. The analyses of IgG subclasses can be useful in monitoring the immunologic response and promoting relevant information in therapeutic conduct. The present study aimed for the identification of
immunoglobulin isotypes in the IgG class associated with the active disease and clinical remission and evaluate the possibility of using the established parameter in prognostic identification. Nineteen children with confirmed diagnostic of visceral leishmaniasis in the Pediatric Service of the Hospital Universitario of UFMS were studied. The serological samples were obtained in three distinct time periods: (P1) before treatment, (P2) one month after treatment and (P3) six months after treatment. The control group was formed by ten healthy children paired according to sex and age. For the ELISA test was used soluble L. chagasi antigen to detection of IgG isotypes (total IgG, IgG1, IgG2, IgG3 and IgG4). The data were expressed in median and standard deviation, considering significant p95%) allowed to group the inhabited localities of the sample and they were contrasted to each other to elaborate a dendogram of epidemic similarity based on the vectorial transmission rate, risk factor determined as more important. Geographically, the populations that inhabit areas with little direct solar illumination independently of the housing type presented more cases (60%) that those that inhabit the areas better illuminated (40%). It was not observed significant differences by altitude for the number of cases located among 790-980 masl. We confirmed that in more than 1300 masl they didn't have Leishmania transmission. The analysis of similarity of a total of 54 compared localities separated three big populations groups that coincide with the geographical location. With molecular tests (PCR) we confirmed in 95% of the cases studied Leishmania (Viannia) braziliensis, as causal agent of the leishmaniasis in the study area. The response to the treatment with diverse systemic therapies for clinic cure demonstrated in the sample that late mucosal engagement doesn't exist. The analysis with PCR-hibrydation demonstrated that the type of treatment neither the time post-cure affects the serological detection of DNA of Leishmania (V) braziliensis. Complement diagnosis ELISA technique didn't evidence seroconvertion in the postcure analyzed cases.
- CONTROL PROGRAMS [492] P 728 - SPATIAL DISTRIBUTION OF VISCERAL LEISHMANIASIS IN THE STATE OF BAHIA, BRAZIL: IMPLICATION FOR ECONOMIC DEVELOPMENT AND CHALLENGES FOR ITS PREVENTION. JÚNIOR, E.R.1; CASTRO, J.M.1; PATEL, B.N.2 1.DIRETORIA DE VIGILÂNCIA EPIDEMIOLÓGICA DA SECRETARIA DE SAÚDE DO ESTADO DA BAHIA, SALVADOR, BA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA, FEIRA DE SANTANA, BA, BRAZIL.
Keyword:epidemiology; bahia; visceral leishmaniasis
Abstract: Visceral leishmaniasis (VL) is a anthropozoonosis disease caused by an obligate intracellular protozoa of the genus Leishmania. It is considered a serious infectious disease, which may present high rate of mortality in endemic areas. The objective of this study is to identify the spatial distribution of LV in Bahia Method: For this purpose, the database” SINAN” of the State Department of Health of Bahia (SESAB), from 2009 to 2011 were consulted.. The degree of LV incidence in the municipalities were classified in to four categories: ‘sporadic’ (meaning less than 2.4 cases of LV during the past three years), “moderate” ( equal to greater than 2.4 cases but less than 4.4 cases of LV), “severe” ( equal to or greater than 4.4 cases of LV) and “silent” (have no reported cases). In Bahia has humid, semi humid and dry climatic regions with different types of vegetation. Results: The data demonstrated a large variation in the incidence of VL in Bahia. From a total of all the 417 municipalities of Bahia, included in the study , 218 (52.27%) presented positive new cases of VL. In 199 (47.72%) of the municipalities no cases of LV were reported, where as 17 (7.79%) of the municipalities were classified in the “ “intense” degree of infestation. In 23 (10.55%) municipalities the LV infection was in the “ moderate “ class. The “sporadic” category of LV infestation was found in 137 (62.84%) of the municipalities. The 17 municipalities with severe infestation were Guanambi, Juazeiro, Salinas da Margarida, Iraquara, Jequié, Cafarnaum, Canarana, Feira de Santana, Irecê, Uibaí, Cabaceiras do Paraguaçu, America Dourada, Sento Sé, Boquira, Camaçari, Barro Alto and Euclides da Cunha. Discussion: We observed that four municipalities, classified as "intense" LV infestation, (Guanambi, Juazeiro, Camaçari and Feira de Santana), present great potential for economic development. These municipalities are undergoing developmental processes that lead to deforestation and the invasion of green areas, followed by intensive irrigated agricultural industry. This situation profoundly alter the ecosystem, favoring the breeding of the leismania vectors and consequent possible transmission of VL, to humans and animals. Conclusion: Taking into consideration the inevitable need for the socio-economic development of Bahia, the current and future challenges are to reconcile this development, taking into account the needs of the public health issues, especially of the visceral Leishmaniasis. There is an urgent necessity for broad and harmonious discussions between the various segments at multi-institutional levels and multidisciplinary segments in Bahia. Public managers( at the state and municipal level) and managers of the agricultural industry, agribusiness, tourism industry, civil construction sector and members of the organized civil society must jointly discuss and develop plans for a sustainable environmental, economic and social development in Bahia. - EPIDEMIOLOGY [496] P 729 - EPIDEMIOLOGY OF VISCERAL LEISHMANIASIS IN THE CITY OF CAXIAS – MA NETO DA SILVA, M.A.C.1; JARDIM FILHO, W.R.2; RIBEIRO, Y.J.S.1; COSTA, E.M.1; CASTELO BRANCO, R.C.1; MUNIZ, C.R.1; DE JESUS, E.A.1; MONTEIRO, E.L.O.1; JUCA, A.A.1; CRUZ, A.S.1; RODRIGUES, G.M.1; DA SILVA, I.S.2; BEZERRA, G.F.B.1; NASCIMENTO, M.D.D.S.B.1 1.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, CAXIAS, MA, BRAZIL.
Keyword:visceral leishmaniasis; epidemiology; caxias - ma
Abstract: Introduction: In Brazil, visceral leishmaniasis (VL) or kala-azar is endemic neotropical, mainly rural, with trends towards urbanization. The etiological agent of VL is Leishmania chagasi, the protozoan intracellular mononuclear phagocytic system. The LV is widely distributed throughout the world. The estimated risk population of 350 million people, currently endemic in 88 countries. In Latin America, Brazil is responsible for 90% of reported cases, 56% in the Northeast. Between 1984 and 2002, 66% of cases occurred in the states of Maranhão, Piauí, Ceará and Bahia. The VL in Brazil affects mainly children, with higher prevalence in males and with the main reservoir Dog From 2006, in Brazil, every case of VL is notifiable, and the data recorded in the Information System Diseases Notification - SINAN. The VT has interrelationships geographic, climatic, social and cultural, which diversifies its clinical manifestations in different regions of the country, as well as being risk factors for the occurrence of the disease. Material and Methods: The study was based on retrospective analysis, cross sectional, descriptive, and quantitative nature documentary of 180 cases of patients diagnosed with visceral leishmaniasis in the period 2007 to 2011, in the city of Caxias - MA. Data were provided by the Department of Epidemiological Surveillance. Were analyzed the following variables: age, sex, race, neighborhood, co-infection with HIV, area of residence, type of entry, education, relationship with work and evolution. Results: We observed that the total of 180 cases (100%), 70% were aged 9 years old, 108 cases were male (60%) and 72 cases were female (40%). As for race, 80.6% were mixed. The neighborhood undertakers was most prevalent (11.25%). Only 3.33% had HIV co-infection. Regarding the area of residence, 88.4% of cases occurred in urban areas, 96.6% were new cases, 43% of cases had incomplete primary education, 3% of the cases were related to work and 92.3% were cured.Conclusions: Visceral leishmaniasis is endemic in the city of Caxias. The council has shown a reduction in the number of cases of illness due to actions of the Municipal Center for Health Education (NMES) with lectures and action of health workers in educating the population, however it still needs more government support in the implementation of most effective measures for the control of VL. - CLINICAL LEISHMANIASIS [498] P 730 - VISCERAL LEISHMANIASIS: LABORATORY AND CLINICAL ASPECTS IN THE CITY OF CAXIAS – MA NETO DA SILVA, M.A.C.1; JARDIM FILHO, W.R.2; RIBEIRO, Y.J.S.1; COSTA, E.M.1; CASTELO BRANCO, R.C.1; MUNIZ, C.R.1; DE JESUS, E.A.1; MONTEIRO, E.L.O.1; JUCA, A.A.1; CRUZ, A.S.1; RODRIGUES, G.M.1; DA SILVA, I.S.2; BEZERRA, G.F.B.1; NASCIMENTO, M.D.D.S.B.1 1.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, CAXIAS, MA, BRAZIL.
Keyword:visceral leishmaniasis; laboratory and clinic; caxias - ma
Abstract: Introduction: Visceral Leishmaniasis (VL) is an anthropozoonosis caused by a protozoan Trypnosomatidae family, genus and subgenus Leishmania and specie chagasi-L. (L.) chagasi. VL is endemic in 88 countries, with 90% of cases occur in Brazil, especially in the Northeast. In VL, the age group below 10 years, especially the smallest of five years, is the most frequently affected. Infection by L. (L) chagasi is characterized by a fever, pallor, weight loss, increased abdominal size, hepatosplenomegaly and edema. It was also observed other clinical manifestations such as cough, diarrhea, jaundice and bleeding that hinder the differential diagnosis with other diseases, delaying the identification, which may cause the patient to death. The diagnosis is made from clinical and epidemiological aspects, and laboratory tests such as complement fixation, indirect immunofluorescence, direct agglutination test, ELISA and DotELISA, and molecular biology techniques such as polymerase chain reaction. The gold standard is the parasitological diagnosis by aspiration of the spleen, bone marrow, liver and lymph nodes.
Material and Methods: A retrospective, descriptive study of diagnosed cases of visceral leishmaniasis in the municipality of Caxias, treated at Children's Hospital Dr. João Viana, in the period 2007 to 2011. The data analyzed were provided by the Department of Epidemiological Surveillance. Were analyzed age, sex, race, diagnostic criteria, clinical manifestations, and initial drug development. Results: Were observed that the total of 180 cases (100%), 70% were aged 9 years old, 108 cases were male (60%) and 72 cases were female (40%). As for race, 80.6% were dun. Regarding diagnostic criteria, there was prevalence of clinical and epidemiological method (85.5%). Regarding clinical manifestations, 6% had bleeding, 21.6% of infections, 51.7% hepatomegaly, splenomegaly 76.7%, pallor 79.7%, 77.9% weight loss, cough and 51.1% / or diarrhea, jaundice 24%, swelling 25.7%, 86.4% and 95 asthenia, fever 5%. The initial drug was antamonial pentavalent in 87.2% of cases. 92.3% were cured. Conclusions: Visceral leishmaniasis is an endemic disease that has many complications, especially in children. Early diagnosis and disease control should be improved VL is a public health problem and must be fought in order to minimize damage and costs. - RESERVOIRS [500] P 731 - STUDY OF CANINE VISCERAL LEISHMANIASIS IN RECENT TRANSMISSION CITY OF MINAS GERAIS STATE, BRAZIL. SOUSA, A.M.1; MENEZES, J.A.2; MORAIS, M.H.G.1; FERREIRA, E.C.3; LIMA, F.P.4; GOMIDE, L.B.5; FONSECA, A.C.6; FILHO, V.P.6; ROCHA, A.M.S.3; FUX, B.7; SOUZA, C.M.2 1.FUNDAÇÃO EDUCACIONAL DE DIVINÓPOLIS, UNIVERSIDADE DO ESTADO DE MINAS GERAIS, DIVINÓPOLIS, MG, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 3.UNIVERSIDADE FEDERAL DE OURO PRETO, OURO PRETO, MG, BRAZIL; 4.PREFEITURA MUNICIPAL DE FORMIGA, FORMIGA, MG, BRAZIL; 5.CLÍNICA VETERINÁRIA ESPAÇO ANIMAL, FORMIGA, MG, BRAZIL; 6.CENTRO UNIVERSITÁRIO DE FORMIGA, FORMIGA, MG, BRAZIL; 7.UNIVERSIDADE FEDERAL DO ESPÍRITO SANTO, VITÓRIA, ES, BRAZIL.
Keyword:canine visceral leishmaniasis; epidemiology; formiga
Abstract: Introduction: Visceral Leishmaniasis (VL) is an insidious disease, with high lethality rate and that urbanized in recent years. In Formiga, Minas Gerais, two autochthonous cases of human VL were reported between to 2011 and 2012, indicating the transmission occurrence in this area. Therefore, the dog being the most important reservoir in large urban centers, the present study aimed to determinate the prevalence of canine disease in the city. Methods: In order to determine the rate of canine infection, are being conducted at random samplings of total blood of domiciled and erring dogs present in the city. This step is being carried out with the assistance of the Municipal Health Department of Formiga, the Center for the Defense of Animal Life (CODEVIDA) and by veterinary clinics in the city. The indirect immunofluorescence (IF) and enzyme linked immunosorbent assay (ELISA) were used to evaluated the prevalence of the disease, these methodology were performed at the Laboratory of Epidemiology of Federal University of Ouro Preto. Parcial Results: The Blood samples were collected from 434 dogs between June to November of 2012. Out of 8.75% (38) were positive, of which 2.53% (11) where positive only by IF and 3.68% (16) only by ELISA and 2.53% (11) of samples were positive by the two tests. The positive dogs by serology 65.8% (25), were classified as asymptomatic, corroborating in studies that most of the dogs infected do not present clinical signs of the disease. When symptomatic, most common symptoms were dermatitis, weight loss and ulcerations. Nineteen samples showed an indeterminate result by ELISA, which may represent an increase in frequency of Canine Leishmaniasis in the city, owing to possible seroconversion. Conclusions: Despite being a city with recent transmission, the rate of canine infection (8.75%) is found into levels above recommended by the World Health Organization (WHO) to implement control measures, which are 2%. Due the fact of VL be able to take on serious forms, is essential to establish preventive measures in the current scenario of the city, where the disease is not considered endemic yet. Thus, this work associated with the study of
sand flies which also comprise this research, would assist in mapping the areas of the transmission risks and the epidemiological surveillance of the disease in the city. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [501] P 732 - SPATIAL DYNAMICS OF L. LONGIPALPIS IN THE CENTRAL REGION OF CITY OF CAXIAS-MA NETO DA SILVA, M.A.C.1; DE SOUSA, M.D.G.2; RIBEIRO, Y.J.S.1; COSTA, E.M.1; CASTELO BRANCO, R.C.1; MUNIZ, C.R.1; BEZERRA, G.F.B.1; NASCIMENTO, M.D.D.S.B.1 1.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, CAXIAS, MA, BRAZIL.
Keyword:visceral leishmaniasis; lutzomya; caxias - ma
Abstract: Introduction: Visceral leishmaniosis (VL) is a anthropozoonosis considered one of the seven major epidemics in the world whose etiological agent is Leishmania chagasi and the vector is the female of phlebotomine Lutzomyia longipalpis, which has been found both in natural ecotopes as in rural and urban. Climatic factors, such as temperature and rainfall, affect the phlebotomine population and the knowledge of seasonality of local species important it´s in planning prevention strategies. To investigate the seasonality of L. longipalpis and the relationship between rainfall and its frequency in a central area of the city of Caxias-MA considering the relevance of this knowledge in endemia´s control. Material and Methods: We performed monthly catches, with CDC light traps in three homes at central area of Caxias Maranhão from September 2007 to June 2009. We used traps inside and outside the houses, exposed from eighteen to six hours. The specimens were identified according to the proposal of Young and Duncan. We used the Spearman test to assess possible correlations between rainfall and frequency of sand flies. Results: We found four species of phlebotomine all from the gender Lutzomyia in a total of 521 insects. The species found were L. lenti, L. evandroi, L. whitmani and L. longipalpis. This last one was the most abundant, occurring predominantly male (94.2%) and more frequently in areas surrounding homes (63.9%) throughout the study period. L. longipalpis was found in all months, except in 2007 (the first month of collection), with a higher frequency in months of rainy season or next to it. There was statistically significant with moderate positive correlations between the events studied, rainfall and monthly number of phlebotomine. Conclusions: The species L. longipalpis was the most frequent and rainfall in the study seems to be a contributing factor to the increase of its population.
- EPIDEMIOLOGY [503] P 733 - VISCERAL LEISHMANIASIS: THE EVOLUTION OF ENDEMIC IN CAXIAS - MA IN THE LAST 8 YEARS NETO DA SILVA, M.A.C.1; DE SOUSA, M.D.G.2; RIBEIRO, Y.J.S.1; COSTA, E.M.1; CASTELO BRANCO, R.C.1; MUNIZ, C.R.1; MONTEIRO, E.L.O.1; BEZERRA, G.F.B.1; NASCIMENTO, M.D.D.S.B.1 1.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL; 2.UNIVERSIDADE ESTADUAL DO MARANHÃO, CAXIAS, MA, BRAZIL.
Keyword:visceral leishmaniasis; epidemiology; caxias - ma
Abstract: Introduction: Visceral leishmaniasis (VL) is a reportable disease, with clinical features of severe outcome, which remains a public health problem. This study aimed to evaluate the epidemiological, clinical, laboratory aspects and treatment of the visceral leishmaniasis,
identifying the socio-demographic aspects of the disease, raising the specific laboratory tests used for diagnosis of VL and deaths during the study period, as well as investigating the situation of canine visceral leishmaniasis in the municipality of Caxias, Maranhão. Material and Methods: We analyzed the records of the Information System for Notifiable Diseases (SINAN) through the Department of Epidemiological Surveillance of the Health Department of the Municipality of Caxias, bank information DATASUS and Center for Zoonoses (CCZ) in Caxias, Maranhão, since January 2003 to May 2010 with a convenience sample. Results: During the period in question were observed 333 human cases of VL, while 10 cases resulted in death (mortality 3.0%). The annual distribution of cases was 77 cases in 2003, 37 in 2004, 54 in 2005, 21 in 2006, 47 in 2007, 42 in 2008, 34 in 2009 and 21 cases registered till May 2010. Most cases occurred in children aged between 1 and 4 years, 143 cases (42.9%), but other age groups were also affected. Prevalent among males with 56.7% of cases and observed that the LV in the city of Caxias is predominantly urban. Most patients had parasitological confirmation, the clinical and epidemiological criterion alone was not relevant. The pentavalent antimony was the drug of first choice in the first treatment in 93.7% of cases. In canine serological surveys conducted in the period 2005 to 2009, 47.3% of canine serum collected was positive. Conclusions: There were no deaths in the last three years, meaning low severity of disease in the city, suggesting that the symptoms and factors associated with unfavorable clinical course of patients with VL in Caxias been prevented and / or addressed at an early stage, reflecting the good quality assistance in the municipality of Caxias. It is noteworthy that still makes it necessary to raise awareness of the government, especially in regard to controlling the transmission. - EPIDEMIOLOGY [504] P 734 - GEOGRAPHIC DISTRIBUTION OF CUTANEOUS LEISHMANIASIS (CL) IN THE PERIOD FROM 2008 TO 2012 IN THE CITY OF BARREIRINHAS, MARANHÃO, BRAZIL. FILHO, A.A.P.1; FONTELES, R.S.2; BANDEIRA, M.C.A.2; MORAES, J.L.P.2; SILVA, S.O.1; REBÊLO, J.M.M.2; MELO, M.N.1 1.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 2.UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL.
Keyword:cutaneous leishmaniasis; geographic distribution; barreirinhas
Abstract: The CL is caused by protozoa of the genus Leishmania, transmitted by sand flies diptera family Psychodidae. In Brazil, the cases are related mainly to the environmental degradation (such as deforestation), lack of investment in education and health infrastructure. Inserted in this context, Barreirinhas, located in Maranhão, has the most of the cases of CL in the state, about 40%. It is related with the tourism, the main economic activity of the region, developed in a predatory way and without proper planning. In addition, the massive deforestation in rural areas, due to the migration of families to these areas, contributes to the increase of cases. Therefore this study aimed to analyze eco-epidemiological aspects of LTA in the villages of Barreirinhas. In 2008, 75 cases were registered with distribution in 39 locations in and around Barreirinhas headquarters, where 08 of the 75 cases occurred in urban areas and the rest in rural areas with the highest concentration in the villages Armazém (04), Baixa D’água (04), Jabuti (06) and Palmeira dos Eduardos (10), this year enrolls approximately 38.26% of the cases. In 2009, the number of positive cases detected in 20 locations was 35, involving new locations such as villages: Pedras, Borges, the district arnaubal (urban area). In 2010, the number of cases suffer a new jump to 61 records of positive LTA involving 35 locations, with the highest concentration in the villages, Piquizeiro (05), Baixa D’Água II ( 06) and Guaribinha (06). In 2011, 48 sites were registered with CL positive cases totaling the highest number since 2008, 81 cases. Between the new affected villages worth mentioning Engenho, Gambá and São Domingos. In the year 2012, the number of cases was 77 distributed in 45 locations, with the highest
concentration in the villages: Fome (10), Barrras (06) and Mangas (05). The city area Barreirinhas is advancing, affecting population growth in the areas of their natural surroundings. In addition, the developmet of agricultural activities in rural areas contributes to degradation of riparian areas and to the promotion of human contact with the vector. Thus, studies about the distribution and dynamics of the vector of this disease endemic in the city and its environments are required. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [505] P 735 - STUDY OF THE PHLEBOTOMINE FAUNA IN FORMIGA, MINAS GERAIS STATE, BRAZIL MORAIS, M.H.G.1; MENEZES, J.A.2; SOUSA, A.M.1; DA FONSECA, A.C.3; FILHO, V.P.3; DILERMANDO, J.A.2; DE SOUZA, C.M.2
1.FUNDAÇÃO EDUCACIONAL DE DIVINÓPOLIS, UNIVERSIDADE DO ESTADO DE MINAS GERAIS, DIVINÓPOLIS, MG, BRAZIL; 2.CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 3.CENTRO UNIVERSITÁRIO DE FORMIGA, FORMIGA, MG, BRAZIL.
Keyword:phlebotomine; ecoepidemiology; formiga (minas gerais state)
Abstract: Introduction: Study of the phlebotomine fauna in foci of recent transmission of leishmaniasis is essential for epidemiological surveillance of the disease, therefore will clarify about the main existing species and their distribution. In Formiga, Minas Gerais, the first case detected of Visceral Leishmaniasis (VL) occurred between 2011 and 2012, however, the micro-region where the municipality is located is endemic for American Cutaneous Leishmaniasis (ACL). Thus, the aim of the present study was to evaluated the local phlebotomine fauna and identify potential areas of the transmission risk. Methods: Monthly, systematic collection of sandflies are being held in the peridomicile of 24 domiciles, using HP light traps for four days consecutively, from 5 p.m. to 7 a.m. The households were selected by convenience in accordance with information from the Municipal Health Department about cases of canine and human Leishmaniasis and the presence of propitious environmental to phlebotomines development. The insects captured are being mounted between slide and coverslip for taxonomic identification using the key of Young and Duncan (1994). Partial Results: From May to October of 2012 were captured 82 phlebotomines of 6 species: 32 Lutzomyia longipalpis (39,02%), 23 Lutzomyia cortelezzi (28,04%), 19 Lutzomyia whitmani (23,17%), 6 Lutzomyia lenti (7,31%), 1 Lutzomyia monticola (1,21%) and 1 Lutzomyia termitophila (1,21%). The first three species were the most abundant during the capture. The highest density of phlebotomines was observed in September, probably due to increased precipitation recorded in that period. The occurrence of VL cases and the gathering of L. longipalpis indicate that, probably, this species is associated with the transmission of the disease in the city, but more studies are required. Furthermore, the incidence of L. whitmani (ACL vector) confirms the necessity of vigilance in the region. Considerations: The encounter of species demonstrably vectors of VL and ACL reinforces the necessity of entomological surveillance in order to prevent the increase of VL cases and the onset of ACL cases in the city. These results associated with the study of canine infection that our group is conducting in the municipality, would give subsidies to map the areas at risk and direct the necessary actions to the control of Leishmaniasis in the region. - EPIDEMIOLOGY [509] P 736 - AUTOCHTHONOUS CASES OF LEISHMANIASIS IN DOGS FROM FLORIANÓPOLIS, SANTA CATARINA, BRAZIL PACHECO, A.D.1; TOMOKANE, T.Y.2; LIMA, V.M.F.1; LAURENTI, M.D.2; MARCONDES, M.1 1.SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARAÇATUBA, SP, BRAZIL; 2.SÃO PAULO UNIVERSITY, SÃO PAULO, SP, BRAZIL.
Keyword:zoonotic visceral leishmaniasis; serology; real-time pcr
Abstract: A total of 491 healthy dogs from the city of Florianópolis, Santa Catarina state, a non endemic area for zoonotic visceral leishmaniasis until 2012, when three cases were first reported, were serologically evaluated by enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFAT) for leishmaniasis. ELISA cut-off value (mean value plus 3 SD) was determined by analysis of serum samples from 30 healthy dogs from a non-endemic area for leishmaniasis. IFAT cut-off value was 1:40. The occurrence of cross-reaction with Ehrlichia canis and Dirofilaria immitis was discharged testing serum samples by a commercial kit (SNAP 4DX, IDEXX Laboratories). Since Santa Catarina state is a non endemic area for Chagas’ disease, free of vector and with no reported case to date, cross-reaction with T. cruzi was also rulled out. Whole blood from dogs with positive results in any of the techniques was also submitted to real-time PCR (qPCR) for Leishmania sp. DNA detection. The population was composed by 337 (68.6%) females and 151 (31.4%) males from 52 (61%) of the 85 neighbourhoods of Florianópolis. Ages ranged from six months to 12 years, with an average of three years. None of the evaluated dogs had visited other counties and all of them were born in Florianópolis. The total seroprevalence of leishmaniasis was 5.3% (25/491); 0.4% (2/491) by ELISA and 4.9% (24/491) by IFAT. In just one dog seroreactivity was observed simultaneously in both serological methods. That was also the only dog in which parasite DNA was amplified in whole blood by qPCR. Seroreactivity occurred in dogs from 14.11% (12/85) of the neighbourhoods of the city. The results of this study confirm the occurrence of autochthonous cases of leishmaniasis in dogs in Florianópolis, and highlight the necessity of an accurate investigation in order to implement control strategies since they can be cases of visceral or cutaneous leishmaniasis. - EPIDEMIOLOGY [516] P 737 - BURDEN OF INFECTION IN COMMUNITIES EXPERIENCING DOMESTIC TRANSMISSION OF DERMAL LEISHMANIASIS IN RISARALDA, COLOMBIA RUBIANO PEREA, L.C.; COSSIO, A.; JOJOA, J.; ROSALES, M.; BLANCO, V.M.; GOMEZ, M.A.; GORE SARAVIA, N. CIDEIM, CALI, COLOMBIA.
Keyword:burden; domestic transmission; prevalence
Abstract: Introduction: Domestic transmission and urbanization of Leishmania Viannia species have become frequent in Colombia and neighboring countries. Contributing factors include population displacement, deforestation and adaptation of sand flies to peridomestic settings. Domestic transmission impacts the most vulnerable population groups: women, children and the elderly. This study sought to determine the age distribution and burden of infection among inhabitants of communities experiencing domestic transmission of cutaneous leishamniasis (CL) Methodology: Age-specific prevalence of symptomatic and subclinical infection was determined in the communities, Itaurí and La Cabaña in Risaralda, Colombia, where sand flies were present within households and children presented healed or active cutaneous leishmaniasis. Prevalence of infection was defined based on clinical and immunological evidence (skin test) and parasitologically using PCR/Southern blot of kDNA and qRTPCR of 7 SLRNA in blood and mucosal tissues (conjunctiva, tonsils, nasal mucosa). Results: The communities presented a pyramidal population profile with 46% ≤ 20 years of age. Among the 180 inhabitants, 124(69%) participated in the study. Overall prevalence of
symptomatic infection was 70% (n=87, 68% historic; 2% active), asymptomatic infection 8% (n=10) and uninfected 22%, (n=27). Prevalence of symptomatic infection increased from 48% in the first decade of life to 78% by the second decade. Subclinical infection ranged from 3% of 010 year olds to 19% of residents > 50 years of age. Leishmania DNA was detected in blood or mucosal tissues of 50% of participants with history of CL or positive skin test between 11 and 50 years of age (n=85) and 10% of children ≤10 years of age (n=20). Parasite DNA was detected in blood of 37% and mucosal tissue of 21% of participants with symptomatic or subclinical infection. Conclusions: High prevalence of infection was documented in settings of domestic transmission based on clinical, immunological and molecular evidence. Considering that one-time sampling provides a minimum estimate, amplification of Leishmania DNA from at least 50% of residents with prior CL or subclinical infection confirmed persistent Leishmania (Viannia) infection in a large proportion of the community. The burden of infection in these and similar settings presents previously unrecognized challenges to control: incident disease will involve children and reactivation of prior infection.
- RESERVOIRS [519] P 738 - LEISHMANIA (VIANNIA) BRAZILIENSIS IN EQUIDS FROM AREAS ENDEMIC FOR AMERICAN CUTANEOUS LEISHMANIASIS IN SOUTHERN BRAZIL: SEROLOGICAL AND MOLECULAR STUDY THOMAZ SOCCOL, V.T.1; TRUPPEL, J.H.2; OTOMURA, F.3; TEODORO, U.3; MASSAFERA, R.4; DALAGRANA, L.5; CATARINO, C.M.6; COSTA-RIBEIRO, M.C.V.6; COSTA FERREIRA, M.E.7 1.UNIVERSIDADE POSITIVO, CURITIBA, PR, BRAZIL; 2.CENTER FOR ZOONOSIS CONTROL, DEPARTMENT OF HEALTH SURVEILLANCE, MUNICIPAL HEALTH DEPARTMENT, ARAUCÁR, ARAUCÁRIA, PR, BRAZIL; 3.POSTGRADUATE PROGRAM IN HEALTH SCIENCES, MARINGÁ STATE UNIVERSITY, MARINGA, PR, BRAZIL; 4.SURVEILLANCE SERVICE OF HEALTH, MINISTRY OF HEALTH, BRAZIL, JACAREZINHO, PR, BRAZIL; 5.UNIVERSIDADE FEDERL DO PARANÁ, CURITIBA, PR, BRAZIL; 6.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL; 7.GEOGRAPHY DEPARTMENT, MARINGÁ STATE UNIVERSITY (UEM), MARINGA, PR, BRAZIL.
Keyword:leishmania braziliensis; molecular epidemiology; reservoir
Abstract: Cutaneous leishmaniasis (CL) is an endemic disease worldwide, an important zoonosis that presents constant, wide geographic expansion due to human-induced deforestation conducted to increase agricultural and pastoral areas. These environmental changes are creating a favorable scenario for the emergence or reemergence of CL and the possibility of new epidemiological patterns of the disease. In this study we detected the infection of equids with L. (Viannia) braziliensis from southern Brazil, in CL endemic areas. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay. Blood samples were collected and tested by polymerase chain reaction (PCR), and the positive products were sequenced. The results showed that 11.0% (25/227) equids were seropositive for L. (V.) braziliensis and 16.3% (37/227) were PCR positive. anti-L. (V.) braziliensis antibodies were detected in 20 horses, 3 donkeys and 2 mules and the parasite DNA was detected in 30 horses, 5 donkeys and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. (V.) braziliensis. Our results show that equids are infected with L. (V.) braziliensis, therefore they could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the CL cycle.
- EPIDEMIOLOGY [523] P 739 - MORTALITY RELATED TO VISCERAL LEISHMANIASIS AND HIV/AIDS COINFECTION IN BRAZIL, 2000-2010 LIMA, M.S.; MARTINS-MELO, F.R.; ALENCAR, C.H.M.; HEUKELBACH, J.; RAMOS JÚNIOR, A.N. UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Keyword:visceral leishmaniasis; hiv/aids; brazil
Abstract: Visceral leishmaniasis (VL) is considered an opportunistic disease in people with HIV/AIDS. Co-infection may reduce the survival of patients significantly, as VL may modify the progression of HIV/AIDS. On the other hand, HIV-induced immunosuppression facilitates progression of VL. This study describes the epidemiological aspects of deaths related to VL and HIV/AIDS co-infection in Brazil, from 2000 to 2010. We analyzed mortality data obtained from the Mortality Information System (Sistema de Informação sobre Mortalidade - SIM /MS /DATASUS) and included deaths that occurred in which VL (ICD-10: B55.0) and HIV/AIDS (ICD-10: B20-B24) were mentioned in the same death certificate (Declaração de óbito - DO), either as underlying or associated causes of death (multiple causes of death). During this period, the association of VL with HIV/AIDS was mentioned in 230 deaths (7.6% of deaths by VL). AIDS was an underlying cause in 60.9% (140), while VL was the underlying cause in 38.3% (88) of deaths. VL and AIDS were presented as associated causes in another 2 deaths. Predominating characteristics were: male gender (78.7%), brown race/color (56.1%), singles (64.3%), age 30-39 years (41.3%), resident in the Northeast region (43.9%) and from all states in Brazil the state of Minas Gerais has the largest proportion of cases (21.7%). Despite the majority residing in rural municipalities (62.2%), the place of occurrence of deaths was mostly in hospitals (97.3%) of the Brazilian state capitals (73%). The systematic description of the epidemiological characteristics and magnitude of mortality related with VL and HIV/AIDS coinfection reflects the need to adopt special strategies to monitor this group. The ruralization of HIV and urbanization of VL in Brazil cause an increasing geographical overlap, representing an important public health problem. Control measures will require comprehensive and reliable information, and an integration of the surveillance of VL and AIDS is needed, as well as enhancement of surveillance of VL and HIV/AIDS co-infection. We recommend the systematic use of HIV testing in patients with VL.
- EPIDEMIOLOGY [526] P 740 - GEOGRAPHIC DISTRIBUITION OF HUMAN AND CANINE VISCERAL LEISHMANIASIS CASES IN THE CITY OF ARACAJU, SERGIPE, BRAZIL TUNON, G.I.; DE MOURA, T.R.; RANGEL, M.U.; SANTANA, A.P.; DE MELO, E.V.; ARAGAO, M.T.; DOS SANTOS, P.L.; DA COSTA, R.V.; LEITE, M.O.; LIMA, S.S.; ALMEIDA, J.A.; ALMEIDA, R.P. UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU, SE, BRAZIL.
Keyword:geoprocessing; leishmaniasis; spatial analysis
Abstract: The change in the epidemiological profile of visceral leishmaniasis from wild / rural to urban environment is a fact that has been occurring in Brazil’s Northeast region. The use of geoprocessing in conjunction with satellite images are extremely useful tools that can be used in epidemiological studies of endemic diseases transmitted by vectors. Considering that there are
few Brazilian studies of canine visceral leishmaniasis based on geoprocessing techniques, we used this approach to determine the spatial distribution patterns of canine and human cases in the city of Aracaju between 2005 and 2012. The aim was to compare the presence of the disease by neighborhood and the distribuition of nitric oxide resistant Leishmania isolates. The study area comprises the area of the county of Aracaju, which is divided in 37 neighborhoods and is located in the coastal area of the state of Sergipe. We georeferenced 2,060 positive dogs and 110 human cases from 2005 to 2012 from data collected from the Zoonosis Control Center and the UFS University Hospital. The cases were spatialized according to geographical location of the address of each reported case. These data were tabulated and exported to Spring 5.1.5 software to generate thematic maps showing the distribution of leishmaniasis cases by neighborhoods of the city of Aracaju. We observed that the human cases of the city happened in areas with relatively high density of leishmaniasis positive dogs, confirming that the occurrence of canine cases precedes the occurrence of human cases. Most human (18) and canine (676) cases occurred in the southern city neighborhoods of Zona de Expansão and Mosqueiro. Comparing the spatial distribution, we noticed that the neighborhoods most affected were those in peripheral areas with lower population densities, recent occupation, poor urban infrastructure and low income. In other neighborhoods close to downtown area (Olaria, Santos Dumont, America and Jardim Centenário), also showed significant cases of human and canine disease. In three neighborhoods (Santos Dumont, America and Dezoito do Forte) where isolated nitric oxide resistant Leishmania from both human and dogs, fact that may be related to a more aggressive form of the disease. The use of geoprocessing techniques along with relevant information of the study area allowed visualization of the geographical distribution of this disease in Aracaju and further facilitate the implementation of appropriate control measures. - CONTROL PROGRAMS [528] P 741 - PERFORMANCE OF KALA-AZAR SURVEILLANCE IN GAFARGAON SUBDISTRICT OF MYMENSINGH, BANGLADESH RAHMAN, K.M.1; MONDAL, D.2; LUBY, S.P.3; SLEIGH, A.C.1
1.AUSTRALIAN NATIONAL UNIVERSITY, CANBERRA, AUSTRALIA; 2.INTERNATIONAL CENTRE FOR DIARRHOEAL DISEASE RESEARCH, BANGLADESH, DHAKA, BANGLADESH; 3.STANFORD UNIVERSITY, STANFORD, UNITED STATES.
Keyword:kala-azar; surveillance; performance
Abstract: Background: The goal of South Asia kala-azar elimination is to lower annual incidence to less than 1 per 10,000 at the district or sub-district level. In Bangladesh, government passive surveillance generated by monthly sub-district reports is being used to monitor progress with the elimination program. We studied performance of the kala azar passive surveillance system in a highly endemic sub-district of Bangladesh. Methods: We conducted a cross-sectional survey from September 2011 to June 2012 in randomly sampled villages of Gafargaon upazila (subdistrict) of Mymensingh district. We screened for kala-azar cases since January 2010, defining cases as those diagnosed by a qualified health care provider with a laboratory test for kala-azar positive. To cross-check with passive surveillance, those identified by our active screening were searched for in the hospital register of the Gafargaon upazila (sub-district) Health Complex (UHC). Results: Of the 67 kala-azar cases detected by screening, we dropped 6 cases from our analysis because they went to a government health facility outside Gafargaon or joined a clinical trial in Gafargaon. Among the remaining 61 cases, 29 (48%) were reported in the (passive surveillance) Gafargaon UHC patient register and eventually contributed to national kala-azar tallies. This proportion increased from 42% in 2010 to 57% in 2011. Conclusion: Around half of the kala-azar cases occurring in a highly endemic sub-district of Bangladesh were not reported through the national passive surveillance system. Larger samples from regions of different levels of endemicity would help confirm these findings. This will eventually contribute to the population based estimation of kala-azar incidence of the country.
- RESERVOIRS [531] P 742 - THE ROLE OF THE DOG IN AN ENDEMIC AREA OF CUTANEOUS LEISHMANIASIS MARQUEZ, E.S.1; NAVARRO, I.T.2; NABUT, L.B.2; CASTRO, E.A.3; COUBEU, S.B.3; RIBEIRO, M.3; FORDELLONE, M.R.C.4; THOMAZ-SOCCOL, V.3 1.UNIVERSIDADE ESTADUL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL; 2.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA, PR, BRAZIL; 3.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL; 4.UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL.
Keyword:cutaneous leishmaniasis; dogs; reservoir
Abstract: The role of the dog as a reservoir of L. infantum has been confirmed, but the same does necessarily not apply to other species, such as L. (V.) braziliensis, which inhabits the largest geographical area in Brazil. Using parasitological, serological and molecular methods, we investigated the presence of Leishmania in different tissues (skin lesions, intact skin, spleen, liver, lymph nodes, bone marrow and peripheral blood) of naturally infected dogs to elucidate the role of the dog in maintaining the cycle of the parasite. To achieve this, 48 (80%) samples from naturally infected dogs with lesions suggestive of American cutaneous leishmaniasis were evaluated. Twenty-eight dogs (58.3%) were positive for serology and isolation of the parasite in vitro. Eight dogs with positive parasitological diagnosis were euthanized in accordance with the regulations of the Brazilian Health Surveillance Agency. During necropsy, blood and several other tissues were collected for parasite research by tissue culture and PCR. The parasite was isolated in culture medium of skin ulcer samples from all the dogs euthanized. Parasite identification by RAPD-PCR determined that the species was L. (V.) braziliensis. Parasite DNA was screened for in tissue lesion samples, using the PCR technique, and seven (87.5%) dogs presented amplified fragments of 70 base pairs. Whole blood of four out of eight dogs (50%) was positive for parasite DNA. The amplified DNA fragments were submitted to sequencing, which presented 100% similarity with the Viannia complex. Positivity was determined in more than one tissue fragment from each dog. The percentages for the different organs investigated were: 100% parasite isolation in skin lesions, 87.5% in bone marrow samples, 62.5% in liver samples, 50% in lymph nodes and blood samples and 37.5% in spleen and intact skin samples. The best explanation for the presence of the parasite in different organs is that following inoculation of Leishmania by the vector, the parasite is preferentially located in the skin, with hematogenous dissemination occurring after an unknown period, such that the concentration of circulating parasites is lower and, consequently, is also lower in other organs than at the skin lesion site. Since the parasite was identified in blood and tissue and the dogs’ period of survival was greater than 12 years, domestic canines may well be good reservoirs for L. (V.) braziliensis. - EPIDEMIOLOGY [533] P 743 - MOBILITY AND SPATIAL DISTRIBUTION OF CUTANEOUS LEISHMANIASIS IN THE LEGAL AMAZONIA, 2007-2009 VALDES, A.C.; OLIVEIRA, R.M.; SABROZA, P.C. ENSP/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:cutaneous leishmaniasis; ecosistema amazónico; spatial distribution
Abstract: Cutaneous Leishmaniasis (CL) is an endemic disease in Brazil. Its transmission depends on specific features, such as vectors strictly limited to certain environmental factors, diversity of agents and reservoirs. Displays transmission patterns urban and rural, related to the type of work shifts to endemic areas and housing type of infected individuals, which makes it necessary to
know the territory and the flow of people in areas where the disease is inserted for surveillance actions . The aim of this study was to correlate the spatial mobility of people and the spatialtemporal distribution of Cutaneous Leishmaniasis, in Amazonia, in the period from 2007 to 2009, considering the flows from the territory of the municipality of residence to the municipality suspected infection. Descriptive epidemiological study focused on understanding the relationship between mobility and spatial distribution of cases of CL in the Legal Amazonia, identifying areas of epidemiological relevance, according to the municipality of infection and considering flows from the territory of the municipality of residence to the municipality suspected infection. Was identified 09 circuits of production of CL, consisting of 15.01% of the municipalities in the region, with 15.182 cases (39.71%), detection average 46.14 cases per 100.000 inhabitants. and average density 4.40 cases/km2, coinciding with the regions of the circuits when analyzed by municipality of residence, but with reduced quantity of municipalities involved. Regarding flows, 5.891 cases (11.69%) present municipality of residence different from the municipality of infection and identified numerous streams with reduced average cases. These flows usually occurred between neighboring municipalities. Regarding poles difference was observed between the areas considered most important for the disease, according to different criteria. Data analysis using municipality of infection and flows allows people realize the difference the poles of epidemiological relevance when used different criteria. Currently, we use only the municipality of residence. This combined with the other criteria, enables the improvement of surveillance, identification of transmission patterns, new areas of epidemiological relevance, especially those related to work processes, enabling improved disease control. - EPIDEMIOLOGY [538] P 744 - CHARACTERIZATION OF PARAGOMINAS/AP AS A POLE OF EPIDEMIOLOGICAL RELEVANCE FOR LEISHMANIASIS IN LEGAL AMAZONIA, 2007 TO 2009. VALDES, A.C.; OLIVEIRA, R.M.; SABROZA, P.C. ENSP/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:cutaneous leishmaniasis; amazon ecosystem; descriptive epidemiology
Abstract: Introduction: In Brazil, Cutaneous Leishmaniasis is an endemic disease. Specific characteristics are related to its transmission, as vectors strictly limited to certain environmental factors and diversity of agents and reservoirs. Its distribution is also related to the flow of people in the territory, mainly related to shifts in the work process. How important area of concern related to the flow of cases and the distribution of Cutaneous Leishmaniasis was identified municipality Paragominas/AP. Objective: To characterize the city of Paragominas a pole of epidemiological relevance for Cutaneous Leishmaniasis in the Legal Amazonia, according to density of cases within a circuit and flow of cases within the period of 2007/2009. Material and Methods: descriptive epidemiological study focused on identification and characterization of a pole of epidemiological relevance for LT. The identification was made by the high density of cases within a circuit, high number of flows of people living in the city by scrolling up and infecting others with the disease, and residents of other municipalities who became infected in polo study, period 2007/2009. And characterization by the analysis of environmental and economic indicators. Results: Paragominas belongs to the Circuit Tucuruí. The municipality has a geographic position that gives advantages to the flow of production through the Belém-Brasília highway (BR-010). Receives significant amount of people seeking employment opportunities, driven by the mining company VALE, engaged in bauxite mining. It is also a major timber poles Legal Amzonia. There was population growth from 2000-2010 in relation to its upper circuit, but with poor urban structure. About Cutaneous Leishmaniasis, during the period, 119 cases were infected in Paragominas and lived in other cities and other 100 cases were infected in other
municipalities and resided in polo. Conclusion: The environmental indicators indicate an economic and population growth of the city, however, urban development has not kept pace. Regarding the flow of people who become infected with Cutaneous Leishmaniasis, these individuals may be being drawn to the region in search of new job opportunities. At the same time, the precarious structure for fixing within this population generates large output stream to nearby towns in search of other opportunities. - EPIDEMIOLOGY [539] P 745 - EXPANSION OF VISCERAL LEISHMANIASIS IN TRÊS LAGOAS - MS ZUQUE, M.A.S.1; ZUQUE, F.T.S.1; ZUQUE, F.R.S.2; LUCHEIS, S.B.3; EMPKE, A.L.T.4; GIMENEZ, D.S.B.4; PETRONI, T.F.1 1.FACULDADES INTEGRADAS DE TRÊS LAGOAS - AEMS, TRÊS LAGOAS, MS, BRAZIL; 2.UNIVERSIDADE FEDERAL DE MATO GROSSO DO SUL - UFMS / CPCX, COXIM, MS, BRAZIL; 3.AGÊNCIA PAULISTA DE TECNOLOGIA DOS AGRONEGÓCIOS (APTA/SAA), PÓLO REGIONAL CENTRO-OESTE, BAURU, SP, BRAZIL; 4.CENTRO DE CONTROLE DE ZOONOSES DE TRÊS LAGOAS, TRÊS LAGOAS, MS, BRAZIL.
Keyword:urbanization; visceral leishmaniasis; endemicity
Abstract: Introduction: There are numerous difficulties in adopting effective measures in urban centers related to the control of Visceral Leishmaniasis (VL), the proposed measures to control are: application of insecticides, diagnosis and appropriate treatment of reported cases, control of the canine reservoir and euthanasia of seropositive dogs to control the strength of canine infection. Objectives: To describe the behavior of the LV at Três Lagoas / MS (Brazil), through control actions recommended by the Ministry of Health. Methodology: Was analyzed data from sectors that: endemic, epidemiological surveillance and the Center for Zoonosis Control ( CCZ) for the period 2007 to 2011. Results: The first cases of Canine Visceral Leishmaniasis (CVL) in the urban area of Tres Lagoas occurred in 1999 and the diagnosis of Human Visceral Leishmaniasis (LVH) in 2000. Was recorded 266 cases of LVH in the period from 2000 to 2006, and the lethality rate ranged from 6% to 50%. There was a decrease in cases from 2007 to 2012, with a record of 115 cases of LVH and lethality rate ranging from 4% to 25%. Were collected for euthanasia the symptomatic dogs or reactives for LV, with 3302 dogs in 2007, 3551 in 2008, 3681 in 2009, 3440 in 2010, 3774 in 2011. The positivity rate in canine surveys of areas of intense transmission ranged from 29% in 2007 to 8% in 2011. Discussion: Was considered factors responsible for the emergence and maintenance of LV cases in the city: a geographic location that allows communication with endemic centers of the disease, environmental change caused by deforestation that changes in the ecosystem of the city; growth and spread of L. longipalpis currently detected throughout the urban area. The dog seems to be the facilitating factor of urbanization and the LV was observed by the high replacement canine population. Since 2007 the city has stepped up measures to control LV and observed a decrease in LVC in areas of intense transmission, sprayed annually and dogs were reactive collected for euthanasia, but there was an increase in LVC in other areas of the city fact observed by the gathering of symptomatic dogs by CCZ. Conclusion: The results suggest the expansion, urbanization and endemicity of the disease in the city with the appearance of cases in each year of the period studied by varying its intensity. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [551] P 746 - DISTRIBUTION OF LUTZOMYIA LONGIPALPIS CHEMOTYPE POPULATIONS IN SÃO PAULO STATE, BRAZIL. CASANOVA, C.1; HAMILTON, J.G.C.2; COLLA-JACQUES, F.E.3; BRAZIL, R.P.4; SHAW, J.J.5
1.SUPERINTÊNDENCIA DE CONTROLE DE ENDEMIAS, SECRETARIA DA SAÚDE DO ESTADO DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.CENTER FOR APPLIED ENTOMOLOGY AND PARASITOLOGY, UNIVERSITY OF KEELE, KEELE, UNITED KINGDOM; 3.INSTITUTO DE BIOLOGIA, PROGRAMA DE PÓS-GRADUAÇÃO EM PARASITOLOGIA, UNIVERSIDADE DE
CAMPINAS, CAMPINAS, SP, BRAZIL; 4.INSTITUTO OSWALDO CRUZ, FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL; 5.INSTITUTO DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:lutzomyia longipalpis; sex pheromone; são paulo state
Abstract: Information on the geographical distribution, dispersal mechanisms and route tendencies of insect-borne diseases is epidemiologically important. It can help to identify ongoing transmission areas, new risk areas and guide surveillance and control activities. Lutzomyia longipalpis (Lutz & Neiva), the principal vector of Leishmania infantum chagasi (Cunha & Chagas) in the Americas, is a species complex that can be separated according to the type of pheromone. It is still unclear how many members there are in this complex, how they are related and if some are more important vectors than others. Recently, we identified two populations in São Paulo State: a 9-methylgermacrene-B pheromone producing population found in the municipalities of Araçatuba, Dracena, Promissão, and Bauru (located in the western region of the state); and a Cembrene-1 pheromone producing population in the municipality of Espírito Santo do Pinhal, located in the north-eastern region of the state. Both chemotype populations were found in peri-urban areas of São Pedro, located in the central region of the state. Human cases have only been recorded in municipalities located in the western region of the state where L. longipalpis 9-methylgermacrene-B population is collected in large numbers and where the majority of the infested municipalities are located. The remarkable differences between the epidemiological situation and the population size of L. longipalpis in these two regions suggest that 9-methylgermacrene-B may have increased vectorial capacities compared to cembrene-1 populations. The number of municipalities harbouring L. longipalpis and the numbers of canine and human AVL cases are all increasing every year in São Paulo state. The present study updates the geographical and progressional distribution of canine and human AVL and its vector. Also the chemotype of L. longipalpis populations from Presidente Prudente, Adamantina, Osvaldo Cruz, Campinas, Itupeva, Sorocaba, Salto, Socorro, Marilia (and others) is presented, and the possible dispersion routes of the different populations are discussed. - EPIDEMIOLOGY [552] P 747 - LEISHMANIA SPECIES IDENTIFICATION AND LEISHMANIAVIRUS DETECTION ON CLINICAL SAMPLES FROM CUTANEOUS AND MUCOCUTANEOUS LEISHMANIASIS PATIENTS IN RONDÔNIA, WESTERN AMAZONIAN REGION. CANTANHEDE, L.M.1; SILVA JÚNIOR, C.F.2; ITO, M.M.2; CUSTÓDIO, M.G.F.1; ALVES, P.H.1; NICOLETE, R.1; SALCEDO, J.M.V.1; GARRIDO, L.M.3; PESCARINI, J.M.3; KRIEGER, H.3; FERREIRA, R.G.M.1 1.FIOCRUZ RONDÔNIA, PORTO VELHO, RO, BRAZIL; 2.UNIVERSIDADE FEDERAL DE RONDÔNIA, PORTO VELHO, RO, BRAZIL; 3.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:leishmania species; leishmaniavirus; cutaneous and mucocutaneous leishmaniasis
Abstract: The American tegumentary leishmaniasis (ATL) is endemic throughout the Amazon region and the state of Rondonia is the third Brazilian state in number of reported cases, with an average of 1000 new cases per year. In Amazonia, seven different species of Leishmania have been described. Clinical manifestation may range from a cutaneous form to the destruction of the mucosal tissues. The mechanisms that lead to disease progression to the mucosal form in about 10% of patients treated or under treatment remain unclear and might be related with parasite and host genetic factors, immune response and more recently the presence a virus infecting the pathogen, named Leishmaniavirus (LRV). The objective of the present study was to identify the Leishmania species causing ACL in Rondonia, Brazilian western Amazonian region, as well as
verify if the LRV could be detected among studied patients and its possible association with the presented clinical form. Cytology brush cotton swabs were collected from lesions of 137 patients with clinical suspicion of leishmaniasis and stored immediately in 1 ml of RNALater®. All samples were subjected to PCR amplification and species identification by RFLP with primers to the internal transcribed spacer 1 (ITS1), to the heat shock protein (HSP70) as well as kinetoplastide DNA (kDNA) – only for leishmaniasis diagnosis. 121 patients presented positive results for at least one of the molecular tests applied. The most frequent species found using HSP70 was Leishmania braziliensis 73 (60.3%), followed by Leishmnia guyanensis 33 (27.3%), Leishmania shawi 2 (1.7%), Leishmania amazonensis 1 (0.8%) and mixed infection was L. guyanensis and L. braziliensis 3 (2.5%), L. guyanensis and L. amazonensis 1 (0.8%). Species from 8 (4.2%) patients were not identified. The cutaneous form was found on 78 patients, mucous form on 38, both forms on 4, and 1 patient presented the diffuse form. For LRV detection, RNA was extracted from the positive samples and cDNA synthesized for PCR amplification using primers specific for the virus genome. The virus was detected in 57 patients (47.1% - 95%CI 38.1%...56.0%). A border line association (p = 0.074, Fisher’s exact test) was found between the presence of LRV and the form of the lesion (excluding the diffuse and mixed forms patients). Overall, the risk of been positive for LRV among cutaneous form patients was 0.772 (95%CI 0,591…1.007) and among mucous for patients was 1,702 (95%CI 0.992…2.915).
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [558] P 748 - LARVAL BREEDING SITES OF LUTZOMYIA LONGIPALPIS (DIPTERA: PSYCHODIDAE) IN VISCERAL LEISHMANIASIS ENDEMIC URBAN AREAS IN SOUTHEASTERN BRAZIL CASANOVA, C.1; ANDRIGUETTI, M.T.M.1; SAMPAIO, S.M.P.1; MACORIS, M.L.G.1; COLLAJACQUES, F.E.2; PRADO, A.P.3 1.SUPERINTÊNDENCIA DE CONTROLE DE ENDEMIAS, SECRETARIA DA SAÚDE DO ESTADO DE SÃO PAULO, SÃO PAULO, SP, BRAZIL; 2.INSTITUTO DE BIOLOGIA, PROGRAMA DE PÓS-GRADUAÇÃO EM PARASITOLOGIA, UNIVERSIDADE ESTADUAL DE CAMPINAS, CAMPINAS, SP, BRAZIL; 3.INSTITUTO DE BIOLOGIA, UNIVERSIDADE ESTADUAL DE CAMPINAS, CAMPINAS, SP, BRAZIL.
Keyword:breeding sites; lutzomyia longipalpis; são paulo state
Abstract: The scarcity of information on the immature stages of sand flies and their preferred breeding sites has resulted in the focus of vectorial control on the adult stage using residual insecticide house-spraying. This strategy, along with the treatment of human cases and the euthanasia of infected dogs, has proven inefficient and visceral leishmaniasis continues to expand in Brazil. Identifying the breeding sites of sand flies is essential to the understanding of the vector’s population dynamic and could be used to develop novel control strategies.In the present study, an intensive search for the breeding sites of Lutzomyia longipalpis was conducted in urban and peri-urban areas of two municipalities, Promissão and Dracena, which are endemic for visceral leishmaniasis in São Paulo State, Brazil. During an exploratory period, a total of 962 soil emergence traps were used to investigate possible peridomiciliary breeding site habitats such as: leaf litter under tree, chicken sheds, other animal sheds and uncovered debris. A total of 160 sand flies were collected and 148 (92.5%) were L. longipalpis. In Promissão the proportion of chicken sheds positive was significantly higher than in leaf litter under trees. Chicken shed habitats presented the highest density of L. longipalpis in both municipalities: 17.29 and 5.71 individuals per square meter sampled in Promissão and Dracena respectively. A contagious spatial distribution pattern of L. longipalpis was identified in the emergence traps located in the chicken sheds.The results indicate that chicken sheds are the preferential breeding site for L. longipalpis in the present study areas. Thus, control measures targeting the immature stages in
chicken sheds could have a great effect on reducing the number of adult flies and consequently the transmission rate of Leishmania (Leishmania) infantum chagasi. - EPIDEMIOLOGY [562] P 749 - A WHOLE BLOOD IFN- Y / IL-10 RELEASE ASSAY AS A MARKER OF THE CELLULAR IMMUNE RESPONSE TO LEISHMANIA DONOVANI INFECTION IN HEALTHY INDIVIDUALS SINGH, O.P.1; SINGH, A.K.1; SINGH, N.1; GIDWANI, K.1; CHOURASIA, A.1; HASKER, E.2; SINGH, R.P.1; MCLEAN, D.3; JONES, S.L.3; BOELAERT, M.2; SUNDAR, S.1 1.DEPARTMENT OF MEDICINE, INSTITUTE OF MEDICAL SCIENCES, BANARAS HINDU UNIVERSITY, VARANASI, INDIA; 2.DEPARTMENT OF PUBLIC HEALTH, INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 3.CELLESTIS LIMITED, CHADSTONE, VICTORIA, AUSTRALIA.
Keyword:ifn- γ / il-10 release assay ; sla, dat, elisa
Abstract: Background: In an area endemic for visceral leishmaniasis (VL), asymptomatically infected persons play crucial role in transmission of disease. We have recently developed a whole blood IFN-γ / IL10 release assay as a marker of cellular immunity to detect infected and non- infected persons with high accuracy. In the present study, we evaluated this test amongst healthy individuals living in the kala-azar endemic zone in Bihar, India for identification of individuals exposed to leishmania infection but without disease. Methods: We enrolled 13,163 persons from eleven highly endemic villages to identify the incident infected healthy persons as measured by seroconversion with DAT and rK39 ELISA at base line survey and at 12 months interval. Whole blood assay was performed longitudinally for two years amongst seropositive and its matched seronegative controls populations using Soluble Leishmania Antigen (SLA). IFN- γ and IL-10 were determined in antigen stimulated whole blood culture supernatant by conventional ELISA. Subjects were followed up on monthly basis to monitor the progression into disease. Results: Of 13,163 persons only 309 subjects (3.6%) had converted to seropositive on either of the DAT or rK39 ELISA in one year interval. The percentage of persons with an IFN-γ-positive response (> 0.78 IU/mL) was equal in both seropositive (19%) and its control groups (16.3%). Of those IFN-γ-positive persons 63.8% and 60.7% person remain IFN-γ positive over 1 year in seropositive and control groups respectively. The new incidence of IFN-γ positivity in healthy persons was 5.31%. Only one subject developed active disease that had high IFN-γ and IL-10 levels at base line. Conclusion: These findings confirm that SLA based IFN- γ / IL-10 release assay is a promising tool for identification of clinically exposed, immune individuals.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [564] P 750 - SUCEPTIBILITY OF PHLEBOTOMUS ORIENTALIS AND P.BERGEROTI TO INSECTICIDES FROM ETHIOPIA MANAGIDO, M.B.; TSEGAYE, I.; GEBRE-MICHAEL, T. AKLILU LEMMA INSTITUTE OF PATHOBIOLOGY, ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA.
Keyword:p.orientalis,p.bergeroti; insecticides; vector control
Abstract: Phlebotomus orientalis is considered as the vector of Leishmania donovani in Ethiopia. P. bergeroti is a suspected vector of L. major elsewhere in Africa and experimental studies have also proven this possibility. It is also suspected to transmit sandfly fever in Ethiopia. In the past, no effort has been made to control sandfly vectors by the application of insecticide based tools in the country. However, exposure to insecticides used for malaria vector control such as indoor residual spraying of insecticides and long lasting insecticidal nets as well as to agricultural pesticides might be inevitable and induce resistance in sandflies. In order to elucidate the status of insecticide susceptibility of the two species, WHO susceptibility tests were conducted on populations of P.orientalis from three localities, Melka-Worer in the Awash Valley in the east, Libo-Kemkem in the north and Metema in northwest. P.bergeroti was tested from Melka-Worer only. The insecticides included one organochlorine (DDT), one orgnophosphate (malathion) and four pyrethroids (permethrin, lambdacyhalothrin, deltamethrin and alphacypermethrin) and tests were conducted on either F1 or wild caught females. The KDT50, KDT95 and mortality was determined. The results showed that the KDT50 and KDT95 of the pyrethroids to P.orientalis from Melka-Worer and Libo-Kemkem remained under 13 and 23 minutes except lambdacyhalothrin in the latter locality in which 18 and 27 minutes were recorded. The Metema population appeared to be less affected as the values of KDT50 ranged from 21.5-28.9 and KDT95 from 31.7-45.7 minutes. On the other hand, the KDT50 of DDT in all places was from 20 to 27 minutes. In terms of mortality, P. orientalis was greatly susceptible to all insecticides (mortality 98-100%) except for malathion from Melka-Worer in which percentage mortality was 93.8. Similarly, P.bergeroti was found to be significantly susceptible to all insecticides as mortality was 100%. However, the KDT50 and KDT95 values of the pyrethroids were greater than those of P.orientalis. In conclusion, both species are susceptible to the tested insecticides, and therefore, vector control in peridomestic habitats using one of these insecticides is warranted in Ethiopia.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [566] P 751 - VECTORS OF THE LEISHMANIASES IN ETHIOPIA GEBRE-MICHAEL, T.1; MANAGIDO, M.B.1; MEKURIA, A.H.2; WARBURG, A.3
1.AKLILU LEMMA INSTITUTE OF PATHOBIOLOGY , ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA; 2.. COLLEGE OF HEALTH SCIENCES, ADDIS ABABA UNIVERSITY, ADDIS ABABA, ETHIOPIA; 3.THE KUVIN CENTER FOR THE STUDY OF TROPICAL AND INFECTIOUS DISEASES,THE HEBREW UNIVERSITY-HADASSAH M, JERUSALEM, ISRAEL.
Keyword:phlebotomus ; visceral leishmaniasis, cutaneous leishmaniasis, ; ethiopia
Abstract: Teshome Gebre-Michael1, Meshesha Balkew1, Asrat Hailu2 and Alon Warburg3 1. Aklilu Lemma Institute of Pathobiology , Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia, 2. College of Health Sciences, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia, 3.The Kuvin Center for the Study of Tropical and infectious Diseases,The Hebrew University-Hadassah Medical School Jerusalem 91120, ISRAEL Abstract Information on the vectors of the leishmaniases in Ethiopia is reviewed. To date, there are least 19 species of Phlebotomus belonging to six subgenera, of which at least eight species are proven vectors of various species Leishmania. Two species, P. (Larroussius) orientalis and P. (Synphlebotomus) martini are the principal vectors of visceral leishmaniasis (VL, L. donovani)
in southern endemic areas of Ethiopia, from which the parasite has been isolated and typed. The former species is also the suspected vector in the much larger and most important endemic areas of northern and northwest of Ethiopia, but the parasite has yet to be isolated/detected. P. (S.) celiae and P. (Anaphlebotomus) rodhaini, sympatric with P. martini in the south while, the latter, with P. orientalis in the north are considered secondary vectors since L. donovani has been detected from few specimens of both species in the south, requiring further investigation. The vectors of cutaneous leishmanisis (CL) caused by L. aethiopica and L. major and L. tropica are various. L. aethiopica which is the most important and widespread cause of CL in the Ethiopian highlands (1700-2700masl) is vectored by three species belonging to two subgenera Larroussius (P. longipes and P. pedifer) and Paraphlebotomus (P. sergenti). However, the parasite was isolated and typed from the latter species in a low altitude Rift Valley area (1000 masl) of eastern Ethiopia where human or animal infections are apparently absent. In addition, P. (Pa.) sergenti and P. (Pa.) saevus) have been found vectors of L. tropica, in the eastern Ethiopia (Rift Valley Region), though CL due to L. tropica is so far rare in Ethiopia. P. (Phelobotomus) duboscqi is a vector of L. major (CL) in southern Ethiopia, where the parasite is similarly so far rare in Ethiopia. Parasites have also been isolated/detected from P. (La.) ashfordi and P. (Adlerius) arabicus, but remain unidentified.
- EPIDEMIOLOGY [581] P 752 - LEISHMANIA PARASITE LOAD STUDY BY QPCR: THRESHOLD VALUE FOR ASYMPTOMATIC INFECTION DETECTION IN KALA AZAR ENDEMIC REGION MUZAFFARPUR, IN INDIA. SUDARSHAN, M.; SUNDAR, S. BANARAS HINDU UNIVERSITY, VARANASI, INDIA.
Keyword:leishmania; qpcr; muzaffarpur,india
Abstract: Background- Case under estimation or misdiagnosis can lead to lethal condition in Kala azar. Infection detecting tool should make the distinction between acute disease,cure status and asymptomatic infection. This work represent qPCR as a tool for detection as well as quantification of Leishmania donovani in individuals from endemic region of Visceral leishmaniasis (VL), i.e., Muzaffarpur, Bihar. Material and Method- Peripheral blood collected in citrate tubes from different subjects are patients(n=40), past case history (cure)(n=94) , endemic healthy (EHC)(n=130) as well as nonendemic healthy(40). Leishmania specific kDNA primer is used for parasite quantification. Result- 36.9% positivity is shown by SYBR green based qPCR in endemic healthy. If detected, qPCR show Parasitemia level in endemic healthy as well as past case history of median value of less than 1parasite genome/ml of blood, having range is 0.008-5.49 while in Patients it is 554,610 parasite genome/ml of blood. Conclusion- Parasite load in different samples determine threshold level as near to 5 parasite genome/ml of blood are on the risk of clinical outcome of infection,i.e, symptomatic. This study will be helpful for the assesment of the endemic persons for detection of parasite load and to categorise them for threshold reference values in cryptic infections as asymptomatic to decide their contribution in disease transmission and their further follow up as progressor or non progressor.
- CONTROL PROGRAMS [588] P 753 - EVIDENCE ON INSECTICIDE TREATED BED NETS TO CONTROL VISCERAL LEISHMANIASIS IN THE INDIAN SUB-CONTINENT CHOWDHURY, R.1; BHANDERI, P.K.2; CHOWDHURY, V.3; FARIA, S.1; BOELAERT, M.4; DASH, A.P.5
1.NATIONAL INSTITUTE OF PREVENTIVE AND SOCIAL MEDICINE (NIPSOM), DHAKA, BANGLADESH; 2.ACI FORMULATION LIMITED, DHAKA, BANGLADESH; 3.DHAKA COLLEGE, DHAKA, BANGLADESH; 4.INSTITUTE OF TROPICAL MEDICINE, ANTWERP, BELGIUM; 5.WHO REGIONAL OFFICE FOR SOUTH-EAST ASIA, NEW DELHI, INDIA.
Keyword:visceral leishmaniasis, vector control; insecticide treated nets, indian subcontinent; elimination, phlebotomus argentipes
Abstract: In the Indian sub-continent (ISC), Visceral Leishmaniasis (VL) is caused by Leishmania donovani, and transmitted by Phlebotomus argentipes. Bangladesh, India and Nepal are working for elimination of VL by 2015 and integrated vector management (IVM) is one of the important elements for that. Indoor residual spraying (IRS) is very effective to reduce peridomestic vector density but it requires strict monitoring and supervision which is not easy to ensure by the national programmes. The use of insecticide treated nets (ITNs) is a potential alternative for sand fly control as bed net use is a common practice in the endemic regions. ITNs combine the individual protection of a bed net with the effect of insecticide. We systematically reviewed the evidence available about the effect of bed nets and ITNs on VL in the ISC. A study in Nepal showed an association between regular use of bed net and protection from VL (OR 0.23, P < 0.001) but another study could not find protective effect. A study in Bangladesh showed consistent use of bed nets especially during summer was protective (OR 0.69, P = 0.01). The efficacy of ITNs tested in a study to prevent L. donovani infection and VL in Nepal and India where ITNs significantly reduced P. argentipes density per house by 24.9%. However, there was no significant difference found in the risk of seroconversion over 24 months. A study in ISC, showed in pooled analysis a 43.7% reduction of sand fly density by ITNs and only in Bangladesh IRS and ITNs were associated with a 70–80% decrease of P. argentipes density up to 5 months post intervention. Vector density rebounded by 11 months post-IRS, whereas ITNtreated households (HH) continued to show significantly lower density compared with HHs without intervention. Another study in Bangladesh has evaluated the effect of ITNs and showed reduced sand fly density by 60% at 18 months post-intervention. In summary, the evidence so far points to a marked to moderate effect of ITNs on sandfly density in the ISC, with possibly a more pronounced effect in Bangladesh than in India and Nepal. There is no evidence so far, ITN would have an additional impact on L. donovani transmission in the current context of VL endemic communities in India and Nepal, with high rates of commercial bed net use and IRS of irregular quality. ITNs should be considered as an additional tool in an IVN strategy without neglecting IRS. More research on alternative and effective sand fly control tools is needed. - EPIDEMIOLOGY [597] P 754 - PHLEBOTOMINIC FAUNA AND NATUAL INFECTION BY LEISHMANIA SPP. IN THE MUNICIPALITY OF NOVA MUTUM, MATO GROSSO, BRAZIL THIES, S.F.1; MARIA, A.L.2; MICHALSKY, É.M.3; MIYAZAKI, R.D.4; FERNANDES, C.J.5; DIAS, E.S.3 1.MEDICAL ENTOMOLOGY LABORATORY, SCHOOL OF MEDICAL SCIENCESS, UFMT/MATO GROSSO STATE SECRETARY/SES., CUIABÁ, MT, BRAZIL; 2.MEDICAL ENTOMOLOGY LABORATORY, SCHOOL OF MEDICAL SCIENCESS, UFMT, CUIABÁ, MT, BRAZIL; 3.LEISHMANIASIS LABORATORY, RENÉ RACHOU RESEARCH CENTRE/FIOCRUZ, BELO HORIZONTE, MG, BRAZIL; 4.BIOLOGY AND ZOOLOGY, INSTITUTE OF BIOSCIENCES, UFMT, CUIABÁ, MT, BRAZIL; 5.DEPARTAMENT OF MEDICAL PRACTICE, SCHOOL OF MEDICINE, JÚLIO MÜLLER UNIVERSITY HOSPITAL, CUIABÁ, MT, BRAZIL.
Keyword:leishmania; nova mutum; l. antunesi
Abstract: The municipality of Nova Mutum is situated in the mid-north region of Mato Grosso state, which in recent years has reported cases of American Tegumentary Leishmaniasis (ATL) in humans. In the rural area one specific place (woodland), has presented human cases with common aspects, such as short incubation period and multiple lesions. The objective of this work was to identify phlebotominic fauna and verify natural infection by Leishmania spp. in a rural area of the municipality of Nova Mutum, Mato Grosso. Phlebotamine sand flies were captured every other month from June 2011 to April 2012 using luminous CDC traps between 17:00 and 07:00 the next day for three consecutive nights, arranged at 10 equidistant points, roughly every 100 metres from the edge into the heart of the forest. The captured specimens were identified according to the Young and Duncan’s proposed classification (1994). Some pools containing between 1 and 10 females were formed, separated by species, the trap number and capture date. The Polymerase Chain Reaction (PCR) method was used to extract and detect Leishmania DNA. The results showed that an extremely diverse fauna, with 31 different species captured, one of the Brumptomyia genus and 30 of the Lutzomyia genus. The most abundant species was L. antunesi (45,44%), followed by L. saulensis (20,57%) which were found every month of collection and at every capture point. 73,15% of the specimens were female. The most prolific month for collection was October 2011. Others species of importance in ATL epidemiology were collected, such as L. flaviscutellata, L. whitmani and L. umbratilis. The highest number of captured phlebotomine sand flies occurred in months of the rainy season (October and December), when the relative humidity and temperature were higher. Thirteen pool naturally infected by Leishmania spp. were found, corresponding to a general infection rate of 4,44% (13/293). The L. antunesi and L. ubiquitalis were found to be naturally infected by Leishmania spp. with rates varying from 5,61% (11/196) to 12,5% (2/16), respectively. The natural infection allied to the abundance in the area investigated suggest the participation of L. antunesi and L. ubiquitalis, in the ATL zoonotic cycle in Nova Mutum, Mato Grosso. - EPIDEMIOLOGY [602] P 755 - PHLEBOTOMINIC FAUNA IN THE FEDERAL UNIVERSITY OF MATO GROSSO (UFMT) CAMPUS AND SURROUNDING NEIGHBOURHOODS IN CUIABÁ, MATO GROSSO THIES, S.F.1; MARIA, A.L.2; RODRIGUES, J.S.2; LEITE, H.S.3; RAMOS, S.R.3; GONÇALVES, E.L.4; MIYAZAKI, R.D.5 1.MEDICAL ENTOMOLOGY LABORATORY, SCHOOL OF MEDICAL SCIENCESS, UFMT/MATO GROSSO STATE SECRETARY/SES., CUIABÁ, MT, BRAZIL; 2.MEDICAL ENTOMOLOGY LABORATORY, SCHOOL OF MEDICAL SCIENCESS, UFMT, CUIABÁ, MT, BRAZIL; 3.REGIONAL HEALTH OFFICE OF BAIXADA CUIABANA, SES, CUIABÁ, MT, BRAZIL; 4.ZOONOSES CONTROL CENTRE, CUIABÁ MUNICIPAL HEALTH SECRETARY, CUIABÁ, MT, BRAZIL; 5.DEPARTAMENT OF BIOLOGY AND ZOOLOGY, INSTITUTE OF BIOSCIENCES, CUIABÁ, MT, BRAZIL.
Keyword:leishmaniases; phlebotomine; ufmt
Abstract: Leishmaniases are zoonotic diseases that can afflict man, transmitted through insects of the Lutzomyia genus, denominated phlebotominae, commonly known as sand flies. The disease is manifested in two forms: American tegumentary leishmaniasis, which afflicts the skin and mucous membranes, and visceral leishmaniasis, which afflicts the spleen, liver and other internal organs. The aim of this study was to monitor the presence of phlebotomine sand flies of veterinary medical relevance on the campus of the Federal University of Mato Grosso and to detect their presence in six local neighbourhoods around the university campus. Monitoring was conducted monthly, from December 2008 to December 2010, with the installation of CDC light
traps at six points around the campus for three consecutive nights from 6:00 p.m. until 6:00 a.m. the following day. The surrounding neighbourhoods monitored were Santa Cruz, Morada dos Nobres, Boa Esperança, Jardim das Américas, Jardim Itália and Renascer, employing the same collection technique, from August to October 2010. Following the collections, the insects were prepared and identified in the Entomology Laboratory of UFMT and the State Secretary of Health, according to guidelines set by Young and Duncan (1994). Thirteen species belonging to the Lutzomyia genus and specimens of Brumptomyia were captured on the UFMT campus, totalling 436 insects, of which 83.03% were found to be of veterinary medical relevance, broken down as follows: L. cruzi (267), L. whitmani (87), L. longipalpis (7) and 1 of the longipalpis complex, 66,57% were female and 33,43% male. In the surrounding neighbourhoods, seven species were found belonging to the Lutzomyia and specimens of Brumptomyia, totaling 42 specimens, of which 15 (35,71%) were of veterinary medical relevance; the presence of L. whitmani was detected in the neighbourhoods of Santa Cruz (1), Boa Esperança (7), Morada dos Nobres (1), Jardim das Américas (1) and insects of the longipalpis complex in Boa Esperança (1), Morada dos Nobres (2) and Santa Cruz (1), of which 42,86% were female and 57,14% male. Considering the abundance and diversity of the findings, an active entomological, epidemiological and environmental surveillance program is recommended to ensure early detection of environmental changes that could alter the population density and diversity of the sand flies and any emergence of new cases of leishmaniasis, thus enabling the adoption of preventive and curative measures, when required. - EPIDEMIOLOGY [608] P 756 - ANALYSIS OF THE EPIDEMIOLOGICAL SITUATION OF VISCERAL LEISHMANIASIS IN THE MUNICIPALITY OF ARAGUAÍNA (TO): PERIOD 2005-2009 TOLEDO, C.R.S.; SOBRAL, A.A.; SABROZA, P.C.; CHAVES, S.A.M. ENSP/FIOCRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:visceral leishmaniasis; dissemination pattern; araguaína, epidemiology
Abstract: The objective of this study was to describe the epidemiological situation of human visceral leishmaniasis (VL) in the urban area of the municipalIty of Araguiana (TO) during the period 2005-2009 in order to understand the pattern of cases dissemination in relation with the social organization from the time-space distribution of the disease. The analysis of the human VL spatial and temporal distribution was correlated with the socio demographic situation and the situation of urban infrastructure, in order to characterize and identify the areas of major vulnerability for the occurrence of the disease in the municipality. For the epidemiological characterization of human cases, we used variables included in the Information System for Notifiable Diseases (SINAN) data bank, exclusively relative to habitants of the municipality of Araguiana: sex, age, co-infection HIV/AIDS and outcome. These variables were assessed by using proportions and specific rates. The confirmed autochtonous cases of human VL were georefrenced based on the address found in SINAN data bank. The study used data from sample surveys conducted canines by the Municipal Health Araguaína to estimate the positive canine in the city between 2006 and 2008. During the study period, a sharp increase in the number of confirmed human VL cases (341.6%) was observed in the municipality with a pattern of diffuse peri-urban dissemination occurring, predominantly, in the urban periphery and, secondarily, in areas central/urbanized. We found a predominance of cases in the age class 0-4 years (49.4%), in agreement with the epidemiological pattern observed in the North Eastern region of Brazil. The main symptoms observed were: fever, asthenia and weight loss. From the survey data canines sampling we observed little change in the positivity of the canine VL in the years 2006 (33.51%), 2007 (37.88%) and 2008 (37.12%) even with the gradual increase in the number of samples collected, respectively, 1549, 4728 and 6223. The analyses reinforced the hypothesis that the occurrence of VL is closely related to the situation of social vulnerability.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [612] P 757 - SEASONAL DYNAMICS, RATES OF INFECTION, AND HOST-FEEDING PREFERENCES OF PHLEBOTOMUS PERNICIOSUS IN A NEW FOCUS OF LEISHMANIASIS IN MADRID, SPAIN. JIMÉNEZ, M.1; GONZÁLEZ, E.1; HERNÁNDEZ, S.1; IRISO, A.2; FÚSTER, F.2; MOLINA, R.1
1.UNIDAD DE ENTOMOLOGÍA MÉDICA, SERVICIO DE PARASITOLOGÍA, CENTRO NACIONAL DE MICROBIOLOGÍA, ISCIII, MAJADAHONDA, MADRID, SPAIN; 2.SUBDIRECCIÓN GENERAL DE SANIDAD AMBIENTAL, CONSEJERÍA DE SANIDAD, COMUNIDAD DE MADRID, MADRID, SPAIN.
Keyword:leishmaniasis; sand flies; madrid
Abstract: Since 2010 it has increased the number of cases of both human visceral leishmaniasis and cutaneous leishmaniasis in southwestern Madrid region (Spain). Direct xenodiagnosis of leishmaniasis in hares (Lepus granatensis) from the focus of the disease proved that they are infective to colonized Phlebotomus perniciosus. Further characterization of promastigotes isolated from these sand flies fed on hares demonstrates that these lagomorphs were infected by Leishmania infantum. Surprisingly the prevalence of canine leishmaniasis in the area of the focus is even lower than that detected in the neighboring areas. In a previous entomological survey carried out in the same area in 2011 P. perniciosus was the only one potential vector identified. In order to a better understanding of the new active focus an entomological survey was carried out from May to October 2012 in 4 stations placed neighbouring the urban area and selected according the high number of sand flies collected in 2011 in the same area. Each month 20 sticky traps (20x20 cm) and 2 CDC traps were used during two consecutive nights in every season. CDC traps were replaced every day. The main objective was to establish the phenology of P. perniciosus in the focus and its relationship to the rates of infection of this species. Additionally we attempted to identify the source of the blood ingested by females. A total of 16181 specimens were collected, 7709 sand flies by CDC light traps and 8472 in sticky traps. Taxonomical identification is in progress. For now three sand fly species have been identified: P. perniciosus (85.3%), P. sergenti (0.03%), and S. minuta (14.6%). 735 P. perniciosus female collected with CDC traps in the 4 stations of seasonal study were dissected and 18 of them were found infected with L. infantum (2.45%). Infected females were found in the four stations during June, July, and August. A total of 14 isolates were obtained and characterized as L. infantum. Host-feeding preferences of sand flies in wild habitats is being done by PCR of cytochrome b, sequencing, and subsequent comparison with sequences deposited in the GenBank database using standard nucleotide BLAST searches. The following hosts have been identified for now: hare, rabbit, dog, and human. The data presented here provide significant epidemiological information relative to the spread of human leishmaniasis in the focus. This study was funded by EU grant GOCE-2003-010284 EDENext and Comunidad Autónoma de Madrid, Spain. - EPIDEMIOLOGY [615] P 758 - PHLEBOTOMINE SAND FLY (DIPTERA: PSYCHODIDAE) FAUNA IN THE MUNICIPALITY OF JABOTICATUBAS, MINAS GERAIS STATE, BRAZIL – PRELIMINARY RESULTS LANA, R.S.1; MONTEIRO, É.M.1; SILVA, J.C.F.2; SILVA, F.O.L.1; COSTA, A.S.N.3; DIAS, E.S.1 1.CENTRO DE PESQUISAS RENÉ RACHOU/FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 2.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.SECRETARIA DE SAÚDE, JABOTICATUBAS, MG, BRAZIL.
Keyword:fauna; lutzomyia; jaboticatubas
Abstract: Jaboticatubas is a Brazilian municipality located in the Serra do Espinhaço at 100 km of the capital of the state of Minas Gerais. It is an ecotourism destination since it holds 80% of the total area of the National Park of Serra do Cipó, an ecological sanctuary with rich and diversified fauna and flora. So far, the phlebotomine sand fly fauna there is unknown. Due to recent notifications of human cases of tegumentary and visceral leishmaniases in the municipality we started an entomological survey aiming to describe the local phlebotomine sand fly fauna and to determine their seasonal fluctuation, the natural rate of Leishmania infection, besides identifying the circulating Leishmania in the sand flies infected, if any. Field captures started on May 2012 and are planned to last on April of 2013. HP light traps are mounted for three consecutive nights per month in eight districts of the urban area and two districts in the countryside of Jaboticatubas. In the first six months of our study, a total of 463 sand flies were captured. Seven distinct species were identified under the following percentages: L. cortelezzi (4.3%), L. intermedia (20.9%), L. lenti (2.2%), Lutzomyia longipalpis (12.7%), L. migonei (0.6%), L. pessoai (5.8%) and L. whitmani (52.9%). The high abundance of phlebotomine sand flies species that are proven vectors of leishmaniases accounted for 87.1% of the captured specimens. Work is in progress to achieve the objectives proposed. Financial support – FAPEMIG, CNPq and FIOCRUZ
- RESERVOIRS [618] P 759 - STUDIES ON THE CANINE VISCERAL LEISHMANIASIS IN BELO HORIZONTE, AN ENDEMIC CAPITAL IN THE STATE OF MINAS GERAIS, BRAZIL. MICHALSKY, E.M.1; LARA-SILVA, F.O.1; SILVA, M.A.1; COSTA, A.J.1; FRANÇA-SILVA, J.C.2; GOMES, L.1; LIMA, A.C.1; FIUZA, V.O.3; PAGLIONI, D.N.3; ASSIS, A.P.3; BUSSOLOTTI, A.S.3; FORTES-DIAS, C.L.4; DIAS, E.S.1 1.CPQRR, BELO HORIZONTE, MG, BRAZIL; 2.UFMG, BELO HORIZONTE, MG, BRAZIL; 3.SECRETARIA DE SAUDE, BELO HORIZONTE, MG, BRAZIL; 4.FUNED, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; belo horizonte; prevalence rate
Abstract: The spreading of the visceral leishmaniasis (VL) in Belo Horizonte, the capital of Minas Gerais state, illustrates well the urbanization of the disease. Since 2001, increasing numbers of human cases of VL have been reported in the city, with 277 human cases between 2009 and 2010. The first human cases were preceded by canine cases and the overall prevalence rate of Leishmania infection in dogs was around 7%, in the same period. In the present work we aim to determine the current status of canine VL in two districts of Belo Horizonte, named Miramar and Salgado Filho. Canine surveys were performed from 2010 to 2012 and Leishmania infection was assayed by enzyme-linked immunosorbent (ELISA) and immunofluorescence (IFA) assays. A total of 4,328 dogs were examined with an average of 721 animals per year and district. The prevalence rates of Leishmania infection per year were determined as 8.1% (2010), 3.7% (2011), 2.3% (2012) for the Miramar district and 4.8% (2010), 2.2% (2011), 4.2% for the Salgado Filho district. After necropsy, tissue fragments from the spleen, bone marrow, lymph nodes and skin from 51 seropositive dogs were tested for the presence of Leishmania parasites by nested PCR (LnPCR) targeting the SSUrRNA gene. The overall percentages of positive samples were as follows: 100.0% for the spleen, 80.4% for the bone marrow, 56.9% for the lymph nodes and 68.6% for the skin, respectively. Bone marrow aspirates were seeded in NNN/LIT culture medium and thirty-eight samples (74.5%) showed Leishmania growth. After subculturing, the promastigotes biomass will be submitted to nested PCR (LnPCR). Gel-extracted amplicons from
both bone marrow subcultures and tissue samples will be sequenced aiming to identify the circulating Leishmania species in Belo Horizonte. Financial support: FAPEMIG, FIOCRUZ, Secretaria Municipal de Saúde de Belo Horizonte, CNPq.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [619] P 760 - PHLEBOTOMINE SAND FLIES (DIPTERA, PSYCHODIDAE) COLLECTED IN THE SURUWAHÁ INDIAN RESERVE, AMAZONAS STATE, BRAZIL ASSIS, M.D.G.G.; BARROS, J.A.C.; SHIMABUKURO, P.H.F. FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:amazon; indigenous people; phlebotominae
Abstract: The Suruwahá are a small, isolated indigenous population of approximately 140 individuals living in the southern Amazon. The Suruwahá live on agriculture and fishing, while hunting is the main and most prestigious activity. Since 2010 there has been an increase in the number of American cutaneous leishmaniasis (ACL) cases recorded among the Suruwahá, which seems to be related to the distribution of flashlights to the community by the Fundação Nacional do Índio (FUNAI). The use of flashlights has caused a shift in the hunting activity of the Suruwahá from strictly diurnal to nocturnal, thus increasing their exposure to the sand fly vectors of Leishmania which bloodfeed at dusk/night. Among the Suruwahá, leishmaniasis is called “idu”, which is also the name of a foul smelling forest mushroom, that resembles the shape of the ACL lesion, and is believed to harbor a spirit that throws arrows that cause the disease. The objective of this work is to study the sand fly fauna collected in the Suruwahá Indian Reserve, Tapauá municipality, Amazonas state, Brazil. The sand flies were collected during a training course on leishmaniasis at the FUNAI Suruwahá base in February 2012. Two CDC light traps were placed at hunting tracks and operated for 3 consecutive nights. We collected 374 sand flies (247 males and 127 females). The most common species was Trichophoromyia ubiquitalis (82%), while the remaining specimens were identified as Th. octavioi, Th. melloi and Th. brachipyga. The sand fly species Th. ubiquitalis has a wide geographical distribution in the Amazon and is an anthropophilic species that has been incriminated in the transmission of Leishmania (Viannia) lainsoni in Pará state. Further studies are necessary to assess the diversity of sand flies in this area in order to understand the transmission dynamics of Leishmania among the Suruwahá. Financial support: CNPq grant no. 474506/2012-6. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [620] P 761 - FREQUENCY OF SANDFLIES (DIPTERA: PSYCHODIDAE: PHLEBOTOMINAE) IN ADRIANOPLE, A TRANSMISSION AREA OF AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF PARANA, BRAZIL. GONÇALVES, A.L.1; CASTRO, E.A.1; SILVA, C.P.C.1; MELO, A.L.A.1; DE SOUZA, N.A.2; THOMAZ-SOCCOL, V.1 1.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA, PR, BRAZIL; 2.FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO, RJ, BRAZIL.
Keyword:american cutaneous leishmaniasis; phlebotominae sand flies; paraná
Abstract: American cutaneous leishmaniasis (ACL) represents a serious public health problem, with repercussions on the economy of endemic countries. Unregulated construction and development by human populations increases the risk of peridomestic transmission and characterizes a new transmission profile. The diversity of sandfly vectors and reservoirs of dermotropic Leishmania species is key improving current understanding of the dynamics of disease transmission. To determine the likely time that the population is more susceptible to being bitten and to acquiring ACL, the hourly rate of sandflies (Psychodidae: Phlebotominae) was investigated in October 2012 in the city of Adrianople, an area endemic for Leishmania (Viannia) braziliensis in the State of Paraná, Brazil. Shannon traps were installed in peridomiciles for one night, between 6 pm and 6 am. In this preliminary assessment, 878 insects were collected. The times of greatest frequency of collection of sandflies were between 3 and 4 am (122 insects collected), midnight and 1 am and 5 and 6 am (120), while fewer insects were collected between 6 and 7 pm (04), 7 and 8pm (28) and between 2 and 3 am (24). The most abundant species was Lutzomyia intermedia s.l., 851 examples collected, followed by Lutzomyia fischeri (15) and Lutzomyia migonei (12). Lutzomyia intermedia s.l. was more abundant during the second half of the night, 547 examples collected. This species predominated between midnight and 1 am and between 3 and 4 am. All the examples of Lu. fischeri were collected between 11 pm and 6 am, while Lu. migonei was also collected more frequently during the second half of the night. The abundance of specimens of Lu. intermedia confirms its dominance in the region and its possible role as a Leishmania vector. Lutzomyia fisheri and Lu. migonei should be considered possible vectors, because they have been found parasitized in other endemic areas in Brazil; however, due to their low density, the vector role is secondary. Studies developed in Adrianople strongly suggest the need to adopt policies that involve health education actions, associated with the notion of environmental management and basic concepts concerning the disease as part of a successful integrated program of entomological surveillance and ACL control.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [623] P 762 - SAND FLIES COLLECTED IN THE CAITITU INDIAN RESERVE (DIPTERA: PSYCHODIDAE: PHLEBOTOMINAE), AMAZONAS STATE, BRAZIL SILVA, T.R.R.1; ASSIS, M.D.G.G.2; FREIRE, M.P.1; GONTIJO, C.M.F.2; SHIMABUKURO, P.H.F.2 1.PPG-SSEA, MANAUS, AM, BRAZIL; 2.FIOCRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:amazon; leishmaniasis; vectors
Abstract: Sand flies collected in the Caititu Indian Reserve (Diptera: Psychodidae: Phlebotominae), Amazonas state, Brazil Túllio Romão Ribeiro da Silva1, Mauro Diego Gobira Guimarães de Assis 2, Maíra Posteraro Freire1, Célia Maria Ferreira Gontijo3, Paloma Helena Fernandes Shimabukuro2 1Programa de Pós-Graduação em Saúde, Sociedade e Endemias na Amazônia, Universidade Federal do Amazonas/Instituto Leônidas e Maria Deane/Universidade Federal do Pará; 2Centro de Referência Nacional e Internacional para Flebotomíneos, Centro de Pesquisas René Rachou, FIOCRUZ/MG; 3Laboratório de Leishmanioses, Centro de Pesquisas René Rachou, FIOCRUZ/MG
American cutaneous leishmaniasis (ACL) in Lábrea municipality is mainly associated with activities such as extraction of Brazil nuts, rubber, copaiba oil, among other forest products, and affects both indigenous and non-indigenous populations. Data on ACL in indigenous populations in this area are scarce, and there is even less information on the etiological agents, the non-human animal reservoir hosts maintaining zoonotic/sylvatic transmission, and the sand fly vectors involved. The objective of this work was to study the fauna of sand flies collected in the Caititu Indian Reserve, Lábrea municipality, Amazonas state, Brazil. The insects were collected during seven nights in February 2012, along the Transamazônica road (BR-230). Sand flies were collected, using 10 CDC light traps placed at the Brazil nut collection sites (“piques de castanha”) used by the indigenous people, during the period of nut collection. The insects were identified to species level using morphology, and DNA extraction from pooled females followed by ITS1 PCR were used to detect Leishmania DNA in these samples. A total of 960 specimens distributed in 11 genera and 25 species were collected. The most abundant species were Psychodopygus davisi (21% of collected sand flies), Nyssomyia antunesi (12%) and Trichophoromyia ubiquitalis (8%). All three species have been previously implicated in the transmission of Leishmania parasites: Le. braziliensis and Le. naiffi, Le. lindenbergi and Le. lainsoni, respectively. A total of 524 females distributed in 78 pools were used for the molecular detection of Leishmania, of which 11 (14%) pools were positive. Future DNA sequencing will provide information on the Leishmania species found in the studied sand flies. Our work confirms both the presence of Leishmania and sand fly vector species in the studied area. More studies are necessary to elucidate the role of each species in the transmission of ACL among the indigenous population of Lábrea municipality. Financial support: Projeto Saúde e Condições de Vida de Povos Indígenas na Amazônia, Programa de Apoio a Núcleos de Excelência – PRONEX/FAPEAM/CNPq, Edital 003/2009.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [625] P 763 - SALIVARY ANTIGENS FROM PHLEBOTOMUS ARGENTIPES: IDENTIFICATION AND ISOLATION MARTÍN-MARTÍN, I.; MOLINA, R.; JIMÉNEZ, M. UNIDAD DE ENTOMOLOGÍA MÉDICA, CENTRO NACIONAL DE MICROBIOLOGÍA, INSTITUTO DE SALUD CARLOS III, MADRID, SPAIN.
Keyword:phlebotomus argentipes; salivary proteins; sand fly
Abstract: In the Indian subcontinent visceral leishmaniasis is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly salivary proteins have been highlighted as vaccine candidates as sand fly saliva is involved in parasite establishment. Moreover, salivary proteins are gaining importance as markers of exposure. Therefore, studies on sand fly saliva would be useful to develop epidemiological tools that help to improve control strategies against leishmaniasis. We identified salivary antigens of P. argentipes by coupling two-dimensional electrophoresis and Western blot techniques. The apyrase SP03, the D7 related protein SP10, the antigen 5 SP05, the 33 kDa-like protein SP09, the PpSP15-like SP01, SP02 and SP07 and other proteins without assigned protein family (SP06, SP17, SP20 and SP56) were described as immunogenic proteins. In a later step, a cDNA library constructed with 120 salivary glands from recently emerged P. argentipes was cloned into λTriplEx2 vector and packaged into phage particles (Gigapack III Gold Packaging Extract, Agilent). The amplified library showed a titer of 1.72 x 1012 pfu/ml and a percentage of non recombinant clones of 5.96 as determined by plating the library by infecting log phase XL1-blue cells in the presence of IPTG and X-Gal. Several salivary antigens such as the apyrase SP03 (DQ136150), the yellow protein SP04 (DQ136151) and the 33 kDa-like
protein SP09 (DQ136155) among others were isolated from the cDNA library and successfully cloned into pCR®4-TOPO® vector (Invitrogen). To our knowledge, this is the first identification of antigens present in the saliva of P. argentipes. Moreover, a cDNA library of high titre and percentage of recombinant clones was successfully constructed and will be useful as a source of material for further studies. Several antigens were isolated from the cDNA library and will be further obtained as recombinant proteins to be tested as vaccines candidates or markers of exposure. This study was funded by the Spanish Ministry of Science & Innovation (Project AGL2008-01592) and EU grant GOCE-2003-010284 EDENext. - RESERVOIRS [626] P 764 - SEROEPIDEMIOLOGY OF CANINE VISCERAL LEISHMANIASIS IN THE MUNICIPALITY OF JABOTICATUBAS, AN IMPORTANT ECOTOURISM DESTINATION IN THE STATE OF MINAS GERAIS, BRAZIL - PRELIMINARY RESULTS. LANA, R.S.1; SILVA, J.C.F.2; MONTEIRO, É.M.1; MARTINS, J.C.D.3; COSTA, A.S.N.3; MARCELINO, A.P.4; AVELAR, D.M.4; CARDOSO, F.A.4; COSTA, A.J.1; SILVA, K.M.S.1; DIAS, E.S.1
1.CENTRO DE PESQUISAS RENÉ RACHOU/FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL; 2.INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL; 3.SECRETARIA DE SAÚDE, JABOTICATUBAS, MG, BRAZIL; 4.FUNDAÇÃO EZEQUIEL DIAS, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine; visceral; leishmaniasis
Abstract: Jaboticatubas is a municipality in the Brazilian state of Minas Gerais, with about 17.600 inhabitants. Jaboticatubas is an important ecotourism destination in the state since it holds 80% of the total area of the National Park of Serra do Cipó, an ecological sanctuary with rich and diversified fauna and flora. Taken into account recent notifications of human cases of visceral leishmaniases in the municipality and the epidemiological role played by dogs in the disease cycle, we focused the present study in canine VL. A census survey was performed with domiciled male and female dogs, from different ages and breeds, in five distinct districts of the municipality. Six hundred and sixty six canine serum samples were collected and screened for VL infection by dual-path platform immunochromatography (DPP®). The positive samples were tested by enzyme-linked immunosorbent assay (ELISA) in order to have each result confirmed. Eight-one samples (12.2%) were found positive by DPP® and about 60% of them were confirmed by ELISA. The high incidence of canine VL associated to the reported presence of the main VL vector, Lutzomyia longipalpis, in Jaboticatubas may favor the transmission cycle of the disease in the municipality.
Financial support: Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), CNPq and FIOCRUZ. - EPIDEMIOLOGY [627] P 765 - CANINE INFECTION AND PHLEBOTOMINE SAND FLY FAUNA IN SABARÁ, AN ENDEMIC MUNICIPALITY IN THE METROPOLITAN AREA OF THE CAPITAL OF THE BRAZILIAN STATE OF MINAS GERAIS. MICHALSKY, E.M.1; LIMA, A.C.1; FRANÇA-SILVA, J.C.2; LARA-SILVA, F.O.1; COSTA, A.A.1; BRITO, J.F.1; SOUZA, M.A.1; DIAS, E.S.1 1.CPQRR, BELO HORIZONTE, MG, BRAZIL; 2.UFMG, BELO HORIZONTE, MG, BRAZIL.
Keyword:canine visceral leishmaniasis; lutzomyia longipalpis; sabará
Abstract: Sabará is a Brazilian municipality with 126,269 inhabitants located in the metropolitan region of Belo Horizonte, the capital of Minas Gerais state. The first cases of canine visceral leishmaniases (CVL) in the whole region were notified in the neighborhood of Sabará. In the present study we aim to verify the current status of CVL as well as the phlebotomine sand fly composition and density in eight districts of that municipality. Two thousand dogs were screened for VL through ELISA and IFA. The prevalence infection rates varied from 2.26% to 5.03%, according to the district, with an average value of 4.25%. Fifty seropositive dogs were necropsied and had tissues (spleen, lymph nodes, skin and bone marrow) collected for further analysis. Bone marrow samples from those dogs were also seeded in culture medium NNN/LIT for parasite isolation. We obtained parasite growth in 38 (76%) of them. Work is in progress to identify the circulating Leishmania species in the municipality. Entomological captures were performed for three consecutive nights per month from January 2011 to December 2012. A total of 482 specimens were captured in the two-year period and the species distribution with respective percentages were as follows: Lutzomyia longipalpis (94.81%), L. intermedia (0.20%), L. sallesi (2.07%) and L. whitmani (1.45%). Although the density of phlebotomine sand flies was apparently low with an average value of 20 specimens per month of capture, L. longipalpis was highly predominant in Sabará. The great majority of specimens (76.55%) were captured in the peridomicile. Our next step will be to determine the natural Leishmania infection rates and the parasite infecting species in those sand flies. Financial support: FAPEMIG, FIOCRUZ, CNPq. - CONTROL PROGRAMS [631] P 766 - IMPORTANCE OF VECTOR SURVEILLANCE IN THE DEFINITION OF PUBLIC HEALTH ACTIONS IN THE MANAGEMENT OF AN OUTBREAK OF LEISHMANIASIS IN MADRID REGION IRISO, A.1; JIMÉNEZ, M.2; VÁZQUEZ, M.Á.3; GONZÁLEZ, E.2; TELLO, A.3; VILAS, F.1; FÚSTER, F.1; GONZÁLEZ, D.3; MOLINA, R.2 1.SUBDIRECCIÓN GENERAL DE SANIDAD AMBIENTAL, CONSEJERÍA DE SANIDAD, COMUNIDAD DE MADRID, MADRID, SPAIN; 2.UNIDAD DE ENTOMOLOGÍA MÉDICA, SERVICIO DE PARASITOLOGÍA, CENTRO NACIONAL DE MICROBIOLOGÍA, ISCIII, MAJADAHONDA, MADRID, SPAIN; 3.DEPARTAMENTO ZOOLOGÍA Y ANTROPOLOGÍA FÍSICA, FACULTAD CIENCIAS BIOLÓGICAS, UNIVERSIDAD COMPLUTENSE, MADRID, SPAIN.
Keyword:leishmaniasis; control; madrid
Abstract: At the end of 2010 an outbreak of human leishmaniasis caused by Leishmania infantum was detected by the Epidemiological Surveillance System of the Autonomous Government of Madrid in the south of the region. Both epidemiological and environmental measures were taken: the study of the disease in different domestic and wild animals (reservoirs), the collection of sand flies (vector) and the implementation of control measures and communication to the population. The aim of the vector investigation is to determine the abundance and distribution of sand flies in the area affected by the outbreak, to evaluate the level of risk for the population and to guide the fight against the vector and the population at risk. It is a very complex action due to the size of the affected area: a surface of 70 km2 where more than 500.000 people live. The surveillance was developed in 2011 and 2012 with the use of sticky paper and light traps. In the year 2012, twenty-four sampling areas were established with 120 sticky paper traps placed fortnightly, following the results obtained in 2011. In the areas with more presence of the vector, specific samplings were made to identify breeding places and to design actions of habitat destruction and insecticide application in order to reduce sand fly densities. With the aim of determine the rates of infection and the source of the female blood meals 2 CDC light traps were used in 2 locations in September and October. In 2012 were used 4 CDC traps in a parallel study
and these data are presented in another abstract. The study has shown the role of Phlebotomus perniciosus as main vector in the outbreak. It was present in more than 60% of the captures and showed high densities near the location of the human cases. The average density exceeded 50 sand flies/km2, with more than 100 specimens in some locations. The activity of this species extended from May to early October. There were two peaks of activity, one in July and a second one in September. Regarding the vector control measures taken an estimated reduction in the sand fly activity of more than 50% has been achieved in treated locations. A strategy for the 2013 has been established, based on both the destruction of sand fly breeding habitats and the complementary use of insecticides. - EPIDEMIOLOGY [633] P 767 - COHORT STUDY OF RISK FACTORS FOR THE PERPETUATION OF CANINE VISCERAL LEISHMANIASIS (CVL) IN ENDEMIC AREA IN THE CITY OF BAURU, STATE OF SÃO PAULO, BRAZIL*. TANIGUCHI, H.H.1; SILVA, S.B.A.1; SANTOS, P.A.1; BARBOSA, J.E.R.1; GONÇALVES NETO, J.R.2; BARBOSA, J.A.R.1; ELIAS, C.R.1; HIRAMOTO, R.M.1; TOLEZANO, J.E.1 1.INSTITUTO ADOLFO LUTZ, SÃO PAULO, SP, BRAZIL; 2.CENTRO DE CONTROLE DE ZOONOSES, BAURU, SP, BRAZIL.
Keyword:canine visceral leishmaniasis; risks factors; reservoirs
Abstract: Visceral leishmaniasis (VL) presents itself in the process of expansion and persistence of foci of transmission in different regions of São Paulo urban areas. The aim of the study was to assess the environmental diagnosis and the importance of canine replacement in the Vila Santa Teresinha, Bauru. Previously this community presented records of dogs naturally infected by Leishmania chagasi. A cohort study was carried out between 2008 and 2012, including 180 households with the presence of dogs. Serological surveys occurred each six months for the diagnosis of canine VL. In the first survey (June 2008 ) of 130 dogs examined 8 were (6.2%) infected. After eight inquiries, 484 dogs examined, 12.0% (58/484) were diagnosed positive, 41 (22.8%) households with the presence of one up to five infected animals. It was found that over 80% of the households with infected dogs replace one or more dogs, and about 22.0% of these households comeback to present infected animals in the following investigations, which confirmed the persistence of the conditions for the maintenance of the foci of transmission. The strategy of performing biannual identification and removal canine reservoirs enabled reduction of the prevalence of canine infection in the first 18 months, followed by a recrudescence in follows surveys. We identified environmental risk factors for colonization of vectors of LVC in households: the existence of terrain or yard vegetation cover, shaded areas; animal feces or decaying organic matter, presence of dogs and other animals. The risks were ranked: absent, mild - up to 3 of these factors, and high - with 4 or more factors. Over 90% of households were classified between medium and high risk environment for canine VL. One should appreciate the quest for environmental sanity, and this should be a priority component in the control of canine VL. *Supported by CNPq – Doenças Negligenciadas.
- DIAGNOSIS – EXPERIMENTAL AND CLINICAL [639] P 768 - DIRECT COSTS OF DIAGNOSTIC TESTS FOR HUMAN VISCERAL LEISHMANIASIS IN BRAZIL MACHADO DE ASSIS, T.S.; GUIMARÃES, P.N.; RABELLO, A.
CENTRO DE PESQUISAS RENÉ RACHOU, FUNDAÇÃO OSWALDO CRUZ, BELO HORIZONTE, MG, BRAZIL.
Keyword:direct costs; diagnostic; visceral leishmaniasis
Abstract: Several diagnostic tests have been used in the management of patients suspected of human visceral leishmaniasis (VL) enrolled in health services in Brazil. Although the performance of these tests is target of several studies, economic aspects are rarely included. The aim of this study was to estimate the direct costs of six diagnostic tests available for VL in Brazil: rapid test IT LEISH® (Bio-Rad Laboratories, USA); rapid test Kala-Azar detect® (INBIOS International, USA); direct agglutination test - DAT-LPC (Laboratório de Pesquisas Clínicas, Centro de Pesquisas René Rachou-CPqRR, Brazil); indirect fluorescence antibody test – IFAT (Instituto de Tecnologia em Imunobiológicos Bio-Manguinhos, Brazil); polymerase chain reaction (PCR) using as target the kDNA of Leishmania and microscopical examination of bone marrow aspirate. In the cost-estimates, only the costs of operational nature were included, assuming that infrastructure and equipment are in place. The main sources of information were the producers of diagnostic kits, the prefecture of Ribeirão das Neves (RN - city of Minas Gerais state, Brazil) and CPqRR. All estimates were calculated taking into consideration the 102 patients with clinical suspicion in RN, during 2011, for whom any diagnostic test was performed by the health system of RN (DAT and rapid tests) or CPqRR (IFAT and PCR).The mean time for test performance - from the collection of biological material until the release of the result - of the rapid tests IT LEISH® and Kala-Azar detect® and the DAT-LPC was of 30, 50 and 40 minutes, respectively, while the mean time for the performance of bone marrow aspirate/examination, IFAT and PCR was of 120, 130 and 190 minutes, respectively. The direct costs of these tests were: DAT-LPC - US$6.9, IT LEISH® - US$7.7, Kala-Azar detect®- US$8.1, IFAT - US$26.7, bone marrow examination - US$27.5, PCR - US$ 43.3. In conclusion, DAT was the diagnostic test with lower direct cost, followed by both rapid tests IT LEISH® and Kala-Azar detect®, while the rapid test IT LEISH® is the faster, followed by DAT and rapid test Kala-Azar detect®. Further studies, evaluating the cost-effectiveness of diagnostic tests for VL are been conducted and may help health managers to set up protocols and diagnostic algorithms. Funding: Fundacão de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG, Belo Horizonte, Minas Gerais, Brazil; Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil. - CONTROL PROGRAMS [644] P 769 - ENVIRONMENTAL MANAGEMENT OF AN OUTBREAK OF HUMAN LEISHMANIOSIS IN THE COMMUNITY OF MADRID (SPAIN): PRELIMINARY ACTIONS. MIRÓ, G.1; CARPINTERO, J.2; SEVILLA, S.3; MARTÍNEZ, A.4; FÚSTER, F.5; ORDOBÁS, M.3; DÍAZ, R.2; DE LA FUENTE, S.4; PEROTE, M.2; ARCE, A.3; IRISO, A.5; ESCACENA, C.6; ESTIRADO, A.3; MARINO, E.5; VILAS, F.5 1.DPTO. SANIDAD ANIMAL, FACULTAD DE VETERINARIA (UCM), MADRID, SPAIN; 2.SUBDIRECCIÓN GENERAL DE RECURSOS AGRARIOS, CONSEJERÍA DE SANIDAD, COMUNIDAD DE MADRID, MADRID, SPAIN; 3.SUBDIRECCIÓN GRAL DE PROMOCIÓN DE LA SALUD Y PREVENCIÓN, CONSEJERÍA DE SANIDAD, COMUNIDAD DE MADRID, MADRID, SPAIN; 4.SERVICIO DE SALUD PÚBLICA AREA 9, CONSEJERÍA DE SANIDAD, COMUNIDAD DE MADRID, MADRID, SPAIN; 5.SUDIRECCIÓN GRAL DE SANIDAD AMBIENTAL, DIRECCIÓN GRAL DE ORDENACIÓN E INSPECCIÓN, CONSEJERÍA SANIDAD, MADRID, SPAIN; 6.SUDIRECCIÓN GRAL DE SANIDAD AMBIENTAL, DIRECCIÓN GRAL DE ORDENACIÓN E INSPECCIÓN, CONSEJERÍA SANIDAD, MADRID, SPAIN.
Keyword:leishmania infantum; human leishmaniosis; outbreak
Abstract: Background: Leishmaniosis due to Leishmania infantum, is a zoonotic disease, which is endemic in Spain, and has been notifiable when found in humans in the Community of Madrid since 1997. In the past decade, between 12 and 25 cases per year have been reported in the region. From July 2009 to December 17, 2012 a total of 403 cases have been reported in the southwest area of Madrid with a corresponding incidence rate of 23.21 per 100,000 people. This communication describes the environmental steps taken to control the outbreak. Methodology: Surveillance activities, research, environmental and epidemiological monitoring coordinated from the Ministries of Health and Environment. The College of Veterinarians of Madrid participated and advice was provided by the Instituto de Salud Carlos III, VISAVET Centre, the Veterinary Faculty and the Faculty of Biological Sciences (Universidad Complutense de Madrid). Results and Discussion: When the outbreak was detected multiple measures were implemented with the following results: - Reservoir: The low seroprevalence in dogs detected using IFAT (less than 2%) does not explain the human outbreak in this area. An abundant hare population has been detected in the affected area and their possible involvement has been examined. Other reservoirs (rabbits and cats) have also been studied. Xenodiagnosis studies confirmed that the hares were acting as a reservoir (29% of positivity by PCR from skin and spleen biopsies in March 2012), and a Lagomorph Control Plan was launched. A total of 1100 hares and 120 rabbits were captured. - Vector: Phlebotomus perniciosus was identified in 64% of the over 20,000 specimens collected and high densities of this insect were detected in the outbreak area (45.3 flebotomines/m2 on average). A Vector Control Plan was also launched and included disinsection, cleaning, clearing, etc. - Environment: Recent environmental changes in the area with the creation of playgrounds, a new road network, etc. have had an impact on the ecology of the sandfly. A plan was set up to establish environmental sanitation measures to eliminate vector habitats, including the cleaning of organic waste, burrows, sewers, etc. - Communication: creation and distribution of information, participation in talks aimed at professionals and people in the area. Conclusions -This is the first report of a non-vectorial transmission route for Leishmania infantum infection involving a new reservoir, the lagomorphs, coinciding in an area with a high density of sandflies. Xenodiagnosis test confirms their active role in the transmission cycle.
- EPIDEMIOLOGY [653] P 770 - SOCIO-ECONOMIC FACTORS ASSOCIATED TO THE OCCURRENCE OF CANINE INFECTION BY LEISHMANIA INFANTUM CHAGASI IN TERESINA – PIAUI – BRAZIL MACEDO, E.C.1; NEGREIROS, A.S.1; WERNECK, G.L.2; SILVA, K.M.1; SILVA, K.R.3; CRUZ, M.D.S.P.1 1.UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL; 2.UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, RIO DE JANEIRO, RJ, BRAZIL; 3.CCENTRO DE PESQUISA GONÇALO MONIZ – FIOCRUZ, SALVADOR, BA, BRAZIL.
Keyword:socio-economic factors; canine; leishmaniasis
Abstract: American visceral leishmaniasis (AVL) is a zoonosis caused by protozoan parasites of the Leishmania genus. In Brazil, the disease is caused by Leishmania (infantum) chagasi and the main vector is the phlebotomine sandfly Lutzomyia longipalpis, found both in natural ecotopes and in the rural and urban environments. This vector is known to adapt easily to the peridomestic environment. The domestic dog has been incriminated as the main reservoir of the parasite in the urban environment, but the control measures based on culling seropositive dogs have not shown to be effective to contain the spread of the disease throughout the country. Many studies evaluated risk factors for human visceral leishmaniasis but few focused on the socioeconomic and environmental factors associated with infection among dogs. Knowledge of these factors might help identify the conditions that contribute to the maintenance of transmission cycles in the urban environment and identify new targets for intervention. The objective of this study was to assess the association between socioeconomic and environmental factors and the occurrence of canine leishmaniasis at Teresina city, Brazil. This cross-sectional study was developed in ten districts of Teresina, involving 536 houses and 800 dogs. Peripheral blood samples were collected by vein punction using vacuntainer tubes without anticoagulant for performing serological tests (indirect immunofluorescence test (IFAT) and Enzyme linked immunosorbent assay (ELISA)). Serum samples were considered positive when positive in both tests. Owners of the selected dwellings were interviewed using a semi-structured questionnaire addressing socioeconomic and environment aspects. The association between variables and seropositivity was assessed through multilevel logistic regression models. Global seropositive prevalence was 42%. There was no statistically significant difference between infection prevalence and age and sex of animals, literacy of the household head, presence of other domestic animals or with household characteristic like water supply, inadequate sewage disposal system, type of floor and roof. Non-breed dogs and those living in houses with absence of masory walls, presence of a kennel, and with longer time of dog residency showed higher odds of seropositivity. These results suggest that some peridomestic characteristics, especially the absence of barriers that allow dogs to have a free access to the street, in association to the presence of a kennel might contribute to maintain the infection cycle between dogs. Intervention measures oriented to the management of the peridomestic environment and responsible dog possession could be useful tools for reducing disease burden in endemic area. - CLINICAL LEISHMANIASIS [654] P 771 - LEISHMANIA INFANTUM CHAGASI AMASTIGOTES IN CEREBROSPINAL FLUID OF A NATURALLY INFECTED DOG CARDINOT, C.B.1; ALMEIDA, B.F.1; LAURENTI, M.D.2; MARCONDES, M.1 1.SCHOOL OF VETERINARY MEDICINE, SÃO PAULO STATE UNIVERSITY, ARACATUBA, SP, BRAZIL; 2.UNIVERSITY OF SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Keyword:zoonotic visceral leishmaniasis; central nervous system; qpcr
Abstract: Visceral leishmaniasis is considered a multisystem disease, and occasionally neurological signs have been observed in naturally infected dogs. There are even few reports of histopathological lesions in central nervous system of asymptomtic dogs, suggesting that parasites or immmunemediate mechanisms are involved in the development of cerebral leishmaniasis. Although amastigotes have been found in choroid plexus, meninges and central nervous system, only Leishmania sp. DNA has been detected in cerebrospinal fluid. A 3-year-old male mongrel dog was referred to São Paulo State University in Araçatuba, an endemic area for zoonotic visceral leishmaniasis, presenting poor nutritional state, pale mucous membranes, enlargment of palpable lymph nodes, hepatosplenomegaly, generalized desquamative dermatitis, onychopathy, corneal ulcer, joint swellings and lameness. Visceral leishmaniasis was confirmed by cytologic diagnosis and the dog was classified in stage C of the disease. In compliance with a federal law the dog was euthanized. Before euthanasia, under general anesthesia, cerebrospinal fluid (CSF) was collected by cerebellomedullary cisternal puncture. CSF analysis revealed a clear appearance; 13 cell/μl with a predominance of small mononuclear cells (66%), followed by large monunucleares (30%) with some macrophages presenting amastigotes of Leishmania, and intact neutrophils (4%); proteins: 38.7 mg/dL. Although the dog had no neurological signs, because of the presence of amastigotes in CSF, samples from meninges, frontal cortex, thalamus, cerebellum, choroid plexus of lateral ventricles and fourth ventricle were collected and subjected to Real-time PCR for Leishmania DNA amplification. Mean parasite load was 16.964 parasites/mL. In none of the previously reported leishmaniasis neurological cases amastigotes were found in CSF and parasite load in CNS was evaluated. These findings demonstrate that parasites reached central nervous system and even without neurological disorders, If this dog had not been euthanized neurological signs would have probably been developed. Some authors state that the use of anti-Leishmania drugs could led the parasite to escape from the drug in the CNS, however, this dogs had not been treated with any drug. - DIAGNOSIS – EXPERIMENTAL AND CLINICAL [659] P 772 - THE FORGOTTEN DISEASE OF CATS: A CASE OF FELINE LEISHMANIOSIS BASSO, M.A.1; MARQUES, C.S.1; DUARTE, A.1; PISSARRA, H.1; CARREIRA, L.M.1; VALÉRIOBOLAS, A.2; SANTOS-GOMES, G.2; TAVARES, L.1; FONSECA, I.P.1 1.CENTRO INTERDISCIPLINAR DE INVESTIGAÇÃO EM SANIDADE ANIMAL, FACULDADE DE MEDICINA VETERINÁRIA, LISBON, PORTUGAL; 2.CENTRO DE MALÁRIA E OUTRAS DOENÇAS TROPICAIS, INSTITUTO DE HIGIENE E MEDICINA TROPICAL, LISBON, PORTUGAL.
Keyword:leishmaniosis; cat; portugal
Abstract: A 2-years-old, domestic shorthair, male neutered cat, was referred in July 2012 to the Veterinary Teatching Hospital of Veterinary Faculty of Lisbon with fever, multiple nodular lesions located in the ears and head and contaminated ulcers in the tibio-tarsical and carpical regions. Since 2011 the cat has been observed by other veterinarians wich treated the animal for pyodermatitis with prednisolone, amoxycillin/clavulanic acid followed by doxycycline without clinical improvement. Following the clinical exam, blood was collected for determination of hematological and biochemical parameters, a fine needle aspirative puncture was performed from the skin nodules and the animal was tested for feline immunodeficiency virus specific antibodies (FIV) and feline leukemia virus (FeLV) antigen. Cutaneous nodular aspirates revealed the presence of Leishmania amastigostes by optical microscopy. To complement this finding, the quantification of antibodies anti-leishmania was
obtained by indirect immunofluorescent assay technique (IFI titre>1280) and qPCR using Leishmania infantum specific probes was performed in blood samples to determine parasitic load. Cultures in Schneider's Insect Medium were also made from the aspirated material, being the isolated promastigotes infective for mouse. Treatment was iniciated with 10mg/kg allopurinol twice a day. After 15 days without any clinical improvement, treatment was reinforced with 50mg/kg of injectable antimoniate of Nmetilglucamine (Glucantime®), once a day, for 30 days. The total remission of the skin nodules of the ears and head was achieved with this treatment but, although the healing seemed to be ongoing, the complete closure of the ulcerative lesions located in the hindlimbs was only possible with a surgical approach. The medical follow-up was done every two days, and serological and molecular testing was performed every two weeks until the end of the injectable treatment. Parasitic load was highly reduced as well as IFI titre (1/320). The findings indicate that the use of N-metilglucamine antimoniate in associaton with allopurinol in the prescribed dose was safe and clinical cure was achieved. Taking into account that the prevalence of feline leishmaniosis in cats in Portugal has been increasing in the last years it is crucial to consider this differential diagnosis in dermatological lesions. Funding: Project FCT PTDC/CVT/ 118566/2010; Grant SFRH/BD/778862011 - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [664] P 773 - GEOMETRIC MORPHOMETRY REVEALS DIFFERENCES BETWEEN NORTHERN POPULATIONS OF NYSSOMYIA NEIVAI FROM ARGENTINA: PRELIMINARY RESULTS UTGÉS, M.E.1; DUJARDIN, J.2; SALOMÓN, O.D.1 1.INSTITUTO NACIONAL DE MEDICINA TROPICAL, PUERTO IGUAZÚ, MISIONES, ARGENTINA; 2.MALADIES INFECTIEUSES ET VECTEURS: ÉCOLOGIE, GÉNÉTIQUE, ÉVOLUTION ET CONTRÔLE (MIVEGEC), IRD, MONTPELLIER, FRANCE.
Keyword:cutaneous leishmaniasis; wing shape variation; ecoregion
Abstract: Nyssomyia neivai is the principal vector of Leishmania sp. causing epidemic Cutaneous Leishmaniasis in nine endemic provinces of Argentina. At the regional scale, studying the heterogenity of populations can give clues to develop common or differential control strategies, to study risk potentiality and migratory routes. If phenotypic variation has environmental and/or genetic causes, its study could help to detect local populations with potentially important characters. Geometric morphometry is a relevant tool for quantifying phenotypic variation and to understand its epidemiological importance. To assess differences in shape and size between Ny. neivai populations, we compared wing shape of each sex between individuals from two endemic provinces: Jujuy (24 females, 83 males) and Formosa (70 females, 52 males). In each province we sampled 3 and 5 sites respectively. For Jujuy’s females, data from one more site in Jujuy was available. Because of low sample size in some sites, the comparisons were only made between the two regions. We used 10 landmarks on each wing. We found significant differences in the wing shape of females between regions (p < 0,005). Sites in Jujuy and Formosa are 400 km apart and belong to different ecoregions (Yungas and Chaco Húmedo, respectively). The discrimination between them was not due to their slight size
difference (size contribution= 3%). Their reclassification based on shape showed 78% of correct validated classification. No significant differences in wing shape were found for males. When analyzing centroid sizes between sites, we found significant differences (p < 0,005) between sites in Jujuy that were 80 km apart. The greatest variability was found at a site in Formosa that was near a goat pen (CV = 5,6 %); however, sample size at this site was low (n=8). These are the first results on geometric morphometry of Ny. neivai wings from Argentine populations. Future sampling efforts may help better characterize these sites and other more in other endemic provinces.
- EPIDEMIOLOGY [668] P 774 - OUTBREAK OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL) IN BANDEIRANTES – PARANÁ IN 2007 ROSA CRUZ, M.F.1; ROSA CRUZ, C.F.1; GALATI, E.B.2; MARQUEZ, E.S.1; ROSA CRUZ, M.F.3
1.UNIVERSIDADE ESTADUAL DO NORTE DO PR, BANDEIRANTES, PR, BRAZIL; 2.FACULDADE SAUDE PUBLICA USP SP, SAO PAULO, SP, BRAZIL; 3.UNIVERSIDADE NORTE DO PARANA, BANDEIRANTES, PR, BRAZIL.
Keyword:american cutaneous leishmaniasis; epidemiogical aspects; outbreak
Abstract: Outbreak of American cutaneous leishmaniasis (ACL) in Bandeirantes – Paraná in 2007 Introduction: American cutaneous leishmaniasis (ACL) is a zoonosis that occurs in men and wild or domestic animals. In the state of Paraná, ACL is endemic, out of the 12,304 cases of the disease reported in southern Brazil from 1980 to 2003, 12,220 (99.3%) occurred in the state of Paraná. Objectives: To describe the occurrence of ACL cases in Bandeirantes in 2007, to recognize the socio-economic, cultural and leisure activities, to identify the risk factors, to describe the knowledge of transmission and the means of prevention, transmission and treatment of ACL. Method: A descriptive epidemiological study using data from a questionnaire previously developed and adapted from other epidemiological studies. Results: There were 36 cases of ACL in 2007. Among the 29 individuals interviewed 66% of cases were female and aged over 60 years. There was a predominance of domestic activity (55%) and 25% had completed only the fourth grade of elementary school, 83% had their own house, while 93% are brick with plaster. Most homes have barns, hen houses and pig sties. There are streams in its vicinity and the presence of orchards, 83% of households are connected to sewage networks, 86% have no public service of garbage collection. Regarding frequency in rural areas, 83% of the subjects attend, 41% have relatives and 34% visit relatives at least once a week. Regarding the environment, the majority reported the presence of organic matter and pets. It is observed that the majority of respondents (66%) did not know about ACL and could not inform any form of prevention. Conclusion: The presence of this breakout suggests that there are ecological conditions that would ensure the reproduction and circulation of parasites. The results suggest that transmission of ACL may be occurring in domestic and peridomestic areas. Most of the people interviewed are not aware of the aspects of transmission, symptoms and prevention of ACL, so interventions are needed in the localities, through educational partnerships with health agencies and educational institutions.
- VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [675] P 775 - LUTZOMYIA LONGIPALPIS IS ASSOCIATED TO BOTANICAL FAMILIES IN BRAZILIAN SEMI-ARID REGION SOARES, M.R.A.1; SANTOS, F.F.M.2; LIMA, M.S.C.S.1; COSTA, C.H.N.3 1.CAMPUS AMÍLCAR FERREIRA SOBRAL/ UNIVERSIDADE FEDERAL DO PIAUÍ, FLORIANO, PI, BRAZIL; 2.UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL; 3.INSTITUTO DE DOENÇAS TROPICAIS NATAN PORTELA/UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA, PI, BRAZIL.
Keyword:leishmaniasis; vector; landscape
Abstract: Background: Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in Brazil with wide distribution. The disease is highly endemic in the semi-arid northeast, where L. longipalpis is found in the wild. Sand flies are associated with plants, which can be a clue for the transmission of VL. Methods: Sand flies were collected in 4 different points of Serra da Capivara National Park, in the semi-arid of southeast Piauí State. A botanic inventory of the area was built by using the line method, with the demarcation of 10 parcels of 10m x 5m. The botanic samples were compared to the herbarium of the Federal University of Piauí. Results: The captures revealed 11 species of the genus Lutzomyia and 31 botanical families. L. longipalpis was the most abundant species (65%), present in all collection points. The botanical families Fabaceae, Convolvulaceae, Poaceae, Malpighiaeceae e Euphorbiaceae were the most abundants. The similarity Sorensen coefficient showed that 3 points were the most similar at the fitossociological point of view. In these areas, the proportion of L. longipalpis fluctuates around 67%. There was a dependency correlation (R2 = 0.89) of the proportion of L. longipalpis to the collection points, when compared to other species of sand flies. Conclusions: This study demonstrated the association of L. longipalpis with botanical families in the semi-arid of Brazil where kala-azar is endemic. The finding indicates the need to map the distribution of L. longipalpis according to the flora, as a possible to tool for the control of the disease. - EPIDEMIOLOGY [685] P 776 - AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF PARANÁ: A RETROSPECTIVE EPIDEMIOLOGICAL STUDY FROM 2003 TO 2008 BISETTO JR., A.B.1; LUZ, E.2; THOMAZ-SOCCOL, V.3 1.SESA- PR, CURITIBA, PR, BRAZIL; 2.UFPR, CURITIBA, PR, BRAZIL; 3.UNIVERSIDADE POSITIVO, CURITIBA, PR, BRAZIL.
Keyword:american cutaneous leishmaniasis ; retrospective epidemiological study; leishmania (viannia) braziliensis
Abstract: Our group analyzed medical records of clinical cases reported between 2003 and 2008 as part of our ongoing investigation of American cutaneous leishmaniasis (ACL) epidemiology due to Leishmania (Viannia) braziliensis in the State of Paraná. The spatial and temporal distribution of the disease were assessed and the detection coefficient (DC) was determined, according to county and health region. We also correlated the disease with the climatic and geographic data and vector occurrence. Entomological data analyses were complemented by reports of entomological surveillance of ACL in the State of Paraná, conducted by the Entomology Center of the State Health Department (SESA-Secretaria de Estado da Saúde). To more fully understand the epidemiological features of ACL, knowledge concerning the populations at risk is required. Based on SINAM data (a national healthcare database) collected between 2003 and 2008, we verified a total of 3667 records, 64.44% of which involved autochthones of their municipality of residence. The highest number of cases, 959, and consequently the highest detection coefficient (CD), was determined for 2003: 12.02 cases per 100,000 inhabitants. In the remaining years the number of cases was between 460 and 643. Analysis verified that males
aged 10 or over and whose profession is related to agriculture are the individuals most at risk of contracting ACL. The largest number of cases occurred in regions with a Cfa Köppen climate, at altitudes of less than 600m and rainfall between 1200 and 1800mm³. The species Lutzomyia intermedia s.l. is the most abundant (56.7%) and presents the widest distribution among the municipalities and the mesoregion. Lu. whitmani is the second species (31.8%) regarding prevalence and distribution. Lu. migonei is present in all CL transmission foci, but at low frequencies. These three species represent 92.8% of those of epidemiological importance in the State of Paraná. Parasite species were isolated from patient samples and identified by PCR and RAPD and 60 strains belonged to L. (V.) braziliensis. In order to control the disease, sanitary authorities must develop assistance, prevention and sequel rehabilitation activities. Environmental management and other control actions are only likely to be effective if education and health promotion actions are implemented.
- EPIDEMIOLOGY [686] P 777 - SEROEPIDEMIOLOGICAL STUDIES OF CANINE LEISHMANIOSIS IN ÉVORA (ALENTEJO, PORTUGAL) SCHALLIG, H.D.F.H.1; CARDOSO, L.2; SEMIÃO-SANTOS, S.J.3 1.KONINKLIJK INSTITUUT VOOR DE TROPEN (KIT), ROYAL TROPICAL INSTITUTE, DEPARTMENT OF PARASITOLOGY, AMSTERDAM, NETHERLANDS; 2.DEPARTMENT OF VETERINARY SCIENCES, ECAV, UNIVERSITY OF TRÁS-OS-MONTES E ALTO DOURO (UTAD), VILA REAL, PORTUGAL; 3.CENTER FOR DIAGNOSIS AND RESEARCH IN LEISHMANIOSIS, ICAAM, UNIVERSITY OF ÉVORA, ÉVORA, PORTUGAL.
Keyword:canine leishmaniosis; direct agglutination test (dat); évora, portugal
Abstract: Canine leishmaniosis (CanL) is a systemic chronic condition and severely affected dogs die unless they are adequately treated. Nevertheless, a major proportion of the infected dogs remains apparently healthy and act as carriers of Leishmania, capable of transmitting parasites to the vector, phlebotomine sand flies. Dogs are the main domestic reservoir for human infection where zoonotic leishmaniosis is caused by L. infantum. The present study describes trends in CanL seroprevalence over two decades in the municipality of Évora, covering an area of 1,307.04 km2 in southern Portugal with an average altitude of 300 metres a.s.l. The climate and vegetation are typically Mediterranean, with dry hot summers (32-35°C) and maximum of rainfall in spring and autumn, and mild winters with temperatures rarely going below 5°C. Canine blood samples on filter paper were collected in the years of 1990 (n = 3,614), 1999 (3,563) and 2010 (n = 1,485). The direct agglutination test (DAT) was used for the titration of antibodies specific to Leishmania. The prevalence of DAT seropositive dogs was 3.9% (n = 141), 9.4% (n = 335) and 5.6% (n = 84) in 1990, 1999 and 2010, respectively. The overall seroprevalence was significantly higher in 1999 (9.4%) compared to 1990 (3.9%), but in 2010 a significant decrease in seroprevalence (5.6%) was found compared to 1999. From 1999 to 2010, seroprevalence significantly turned from higher to lower in the contiguous urban areas compared to the contiguous rural ones. Out of the 141 seropositive dogs in 1990, 113 (80.1%) were apparently healthy and 28 (19.9%) were considered clinically suspect (0.8% of the total study population in 1990). Similar observations were made in 1999 and 2010, where 263 healthy dogs (78.5%) and 72 (21.5%) suspect dogs (2.02% of the total study population) were DAT positive and 71 (80.1%) healthy and 13 (9.9%) suspect dogs (0.87% of the total study population) were seropositive, respectively. Lymphadenopathy, followed by onychogriposis and skin involvement, were the most frequently observed clinical signs. A significant number of dogs of Évora are seropositive for leishmaniosis. A peak in seropositivity was observed in 1999 and there was a decline in the number of seropositive cases in 2010, to the levels of 1990. There was a considerably high proportion of dogs that are seropositive but appear as clinically healthy.
Measures to control human zoonotic leishmaniosis should remain focussed on the canine population. - VECTOR BIOLOGY AND CONTROL: EXPERIMENTAL AND FIELD WORK [689] P 778 - COMPARISON OF TWO TYPES OF LIGHT TRAPS TO CAPTURE SAND FLIES: WHITE AND BLACK LED LIGHT. FERNANDEZ, M.S.1; SANTINI, M.S.2; GOULD, I.T.2; MARTINEZ, M.F.3; BERROSPE, P.E.2; MANTECA-ACOSTA, M.4; PEREZ, A.A.5; SALOMON, O.D.3
1.CENTRO NACIONAL DE DIAGNÓSTICO E INVESTIGACIÓN EN ENDEMO-EPIDEMIAS / CONICET, BUENOS AIRES, ARGENTINA; 2.CENTRO NACIONAL DE DIAGNÓSTICO E INVESTIGACIÓN EN ENDEMO-EPIDEMIAS, BUENOS AIRES, ARGENTINA; 3.INSTITUTO NACIONAL DE MEDICINA TROPICAL / CONICET, PUERTO IGUAZÚ, ARGENTINA; 4.INSTITUTO NACIONAL DE MEDICINA TROPICAL, PUERTO IGUAZÚ, ARGENTINA; 5.DPTO DE ECOLOGÍA, GENÉTICA Y EVOLUCIÓN, FACULTAD DE CIENCIAS EXACTAS Y NATURALES, UBA, BUENOS AIRES, ARGENTINA.
Keyword:sandflies; traps; leds
Abstract: In Argentina leishmaniasis is an endemic and epidemic disease that it has expanded its distribution in recent years. To study the presence and abundance of sandflies involved in the transmission of this disease, we have developed an economic and new type of light traps using white-light LEDs (LLW) and black-light LEDs (LLB). The new traps are a modified CDC minilight trap with a leds ring. The aim of this work was to compare the LLW and LLB traps in relation to phlebotominae captures. A total of 542 traps were located in different sites: peridomiciles of households in urban and rural areas and forest environments, placed in four provinces of Argentina. One LLB and one LLW traps were placed in each site overnight (placed on consecutive nights, randomized one trap per night). Only 76 sites resulted positives for phlebotominae captures (41/190 urban, 24/55 rural and 11/26 forest), with a total of 2711 sandflies captured. Nine species where present: Lutzomyia longipalpis (50.8%), Nyssomyia whitmani (29.4%), Migonemyia migonei (17.3%) and the remaining species representing 2.5% of the total captures (Nyssomyia neivai, Psathyromyia shannoni, Evandromyia cortelezzii, Ev. sallesi, Micropygomyia quinquefer, Pintomyia pessoai and Brumptomyia sp.). Positive association was found between the estimated abundance with both wavelengths for both sexes (rs=0.83, p 2000 mg/kg. According to allometric scaling predictions for human PK, the minimum efficacious dose in human beings (60 kg) is expected to be in the range of 200 to 600 mg, with a once a day oral administration. In conclusion, DNDI-VL-2098 so far shows promising properties to be developed as a clinical candidate for the treatment of visceral leishmaniasis. Title: Towards the identification of a back-up candidate for DNDI-VL-2098 to treat visceral leishmaniasis
Author names: Delphine Launaya, Eric Chatelaina, Stéphanie Braillarda, Preeti Vishwakarmab, Suman Guptab, S.K. Purib, Vanessa Yardleyc, Louis Maesd, Andrew Thompsone Author affiliations: a
Drugs for Neglected Diseases initiative, Geneva, Switzerland; bCentral Drug Research Institute, Lucknow, India; cLondon School of Hygiene and Tropical Medicine; dLaboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Belgium; eAuckland Cancer Society Research Centre, University of Auckland, New Zealand. Abstract text The Drugs for Neglected Diseases initiative (DNDi) is a collaborative, patients’ needs-driven, not-forprofit organization whose mission is to develop new drugs for the most neglected tropical diseases, including Visceral Leishmaniasis (VL). Through collaboration with the TB Alliance/Auckland University, DNDi identified DNDI-VL-2098 as a potential drug candidate for VL, and is currently completing a set of preclinical studies. Since nitroimidazoles still hold great potential as treatment for neglected tropical diseases (including VL), the project team decided to focus its efforts on a back-up program. The main objectives were as follows: a) modify the core structure of DNDI-VL-2098 (nitroimidazooxazole) to encompass potential safety liabilities; b) improve pharmacological properties; c) decrease hERG inhibition potential and d) show efficacy in in vivo models. To address these objectives, it was decided to concentrate on substituted nitroimidazooxazine analogues. High-throughput screening of a library of nitroimidazooxazines at IPK (Institut Pasteur Korea) led to a number of hits from two subseries that were progressed into lead optimization. Medicinal chemistry work on those hit series was mainly concentrated on side chain variations, and particularly on incorporating new heteroaryl systems designed to improve the overall pharmacological profile of the current lead. To date, around 230 compounds were synthesized and screened in vitro at CSIR-CDRI against the intramacrophagic amastigote stage of the parasite (VL) expressing luciférase firefly reporter gene. From these, 190 compounds showed IC50 < 1 µM and more than 60 compounds had IC50 < 100 nM. Selected compounds with excellent in vitro potential (potency as well as microsomal stability) were evaluated in vivo. Those compounds with the most encouraging efficacies in both mouse and hamster models of VL are currently being progressed as back-up candidates. Additional studies are in progress to further characterize these and to select a back-up compound with the best chance of becoming a successful clinical candidate.
DEVELOPMENT OF NEW DRUGS FOR LEISHMANIASIS : WHAT CAN WE LEARN FROM THE IN VIVO PROFILING OF SEVERAL PROMISING CHEMICAL SERIES STEPHANIE BRAILLARD1; ERIC CHATELAIN2; CHARLES E. MOWBRAY3; DELPHINE LAUNAY4; LOUIS MAES5; ANDREW THOMPSON6. 1,2,3,4.DNDI, GENEVA - SWITZERLAND ; 5.LABORATORY OF MICROBIOLOGY, PARASITOLOGY AND HYGIENE, UNIVERSITY OF ANTWERP, ANTWERP - BELGIUM ; 6.AUCKLAND CANCER SOCIETY RESEARCH CENTRE, UNIVERSITY OF AUCKLAND, AUCKLAND - NEW ZEALAND . Drugs for Neglected Diseases initiative (DNDi) is a collaborative, patients’ needs-driven, not-for-profit organization whose mission is to develop and make available new drugs for the most neglected tropical diseases, such as visceral leishmaniasis (VL). DNDi’s Lead Optimization programs aim to develop and improve chemical entities that have to fulfill the Target Product Profile (TPP) in terms of efficacy, safety and cost. To this end, building a robust and accurate screening cascade for identifying potential candidates from discovery screening to preclinical development is crucial. This is a dynamic, two-way process that depends on the results obtained through in vivo profiling and, retrospectively, from clinical outcomes. Partnership between DNDi, academic laboratories with leishmaniasis or series specific medicinal chemistry expertise and committed Contract Research Organizations led to the generation of a reasonable number of in vivo data both on pharmacokinetics and efficacy. Efficacy data was obtained through in vivo assessment in the Balb/c mouse model, and more recently in the Syrian hamster model developed at LMPH (Laboratory of Microbiology, Parasitology and Hygiene). This hamster model, which aims to predict results in humans as much as possible, showed robust and highly reproducible results. Supplemented by pharmacokinetic data in different species, this efficacy model allowed us to start building a preliminary assessment of the Pharmacokinetic/Pharmacodynamic (PK/PD) relationships for different new VL chemical series, such as oxaboroles and nitroimidazoles. Moreover, evaluation of existing drugs or new formulated drugs will allow us to benchmark this model, and, more importantly, will reinforce the accuracy of DNDi’s screening cascade through a better understanding of the in vitro / in vivo profiling as a function of the different mechanisms of action.
NEW DRUGS FOR LEISHMANIASIS: FROM SCREENING TO NEW CANDIDATES FOR CLINICAL DEVELOPMENT Charles E. Mowbray Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland The treatment options for patients suffering from visceral leishmaniasis (VL) are limited and are often old drugs with serious limitations due to toxicity, inconvenient routes of administration, long duration of therapy and limited efficacy. There is an urgent need to deliver a new generation of safe, effective and convenient medicines to tackle this disease and combat the threat of drug resistance. A short review of the properties and issues associated with some of the approved drugs for VL will serve to underline the on‐going, unmet medical need for new and improved medicines. However, there are significant challenges to targeting the protozoan Leishmania parasites which are the causative agents of VL. There are few validated anti‐parasitic mechanisms and furthermore the parasites are intracellular presenting additional challenges in delivering drugs to their site of action. At DNDi we have adopted a phenotypic screening approach to identify new chemical leads. We are fortunate to have access to the screening collections of a number of generous partners; however we are faced with a number of challenges in selecting appropriate compounds to most effectively utilize our available screening capacity. Some of the lessons and conclusions from our screening campaigns will be presented. Our screening cascade for optimizing initial hits into optimized leads suitable for pre‐clinical and subsequent clinical evaluation will be described and illustrated with examples of leads from novel chemical classes which present promise for future clinical studies.
Title: Treatment access challenges in leishmaniasis; recommendations Author name(s): Margriet den Boer1, Daniel Argaw1, Ivan Velez2, Jorge Alvar1,3 Author affiliation(s): 1
World Health Organization, Geneva; 2 PECET (Program for the Study and Control of Tropical Diseases), University of Antioquia, Medellin, Colombia; 3 Drugs for Neglected Diseases initiative, Geneva Abstract text In a world-wide survey performed in 2008-2010 in all countries endemic for leishmaniasis, a multitude of treatment access problems were investigated. A detailed analysis of the outcomes is the content of this presentation. Multiple challenges for treatment access are posed by a lack of access to leishmaniasis treatment services. Medicine related factors, such as limited production, high prices, absence of pooled forecasting and procurement, and a less-than-thorough quality supervision have caused regular supply ruptures in the past with serious consequences for programs. Other factors that impede treatment access include lack of National Leishmaniasis Control Programs in the majority of the countries, or inadequate funding to roll them out. Diagnosis and treatment is not always available at primary health care level, and many leishmaniasis-endemic areas are very remote, with no health facilities and poor means of transport. Especially in coetaneous forms, patients will often use traditional medicines based on plants and caustics that are perceived as a better option than treatment offered in health care facilities. Patients are generally extremely poor and suffer economical catastrophe when they spend time away from home in order to receive treatment, or cannot afford treatment at all. There is also a lack of awareness among patients of the serious nature of the disease, causing them to seek substandard private care. Where treatment is decentralized, inadequate trained human resources and discontinuous drug supply pose problems, and in some cases, ineffective means of diagnosis and drugs are offered, not in line with WHO’s recommendations. These problems can be overcome with increased commitment of the governments of endemic countries, development partners, and international donors, and continued cooperation with pharmaceutical manufacturers to make existing treatments accessible and develop new treatments that are safe and affordable. Recently, a significant difference in treatment access has been observed in South Sudan, delivering a proper response to the recent outbreak that affected more than 25,000 patients, in which the reported case fatality rate was
