Leishmania infantum detection in Nyssomyia neivai and dogs in Southern Brazil.

BACKGROUND: The sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak. METHODS: This report includes a survey of the sand fly fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR. RESULTS: A total of 3419 sand flies, belonging to five genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fischeri (0.8%), Evandromyia edwardsi (< 0.1%), and species of the genus Brumptomyia. (0.1%). Out of the 509 non-engorged females analyzed by PCR, two (0.4%) carried L. infantum DNA. The naturally infected females were identified as Ny. neivai, in both morphological and molecular analysis. In addition, two out of 47 conjunctival swabs from dogs tested positive for L. infantum, yielding an infection rate of 4.2%. CONCLUSIONS: These results confirm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil.

Molecular Detection of Leishmania spp. and Blood Source of Female Sand Flies in the Parque Estadual do Rio Doce and Municipality of Timóteo, Minas Gerais, Brazil.

Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania and is transmitted by the bite female sand fly. The present work is characterized as a descriptive study in two areas: a forest area located in the Parque Estadual do Rio Doce, and another urban area located in the municipality of Timóteo-MG, with the objective of identifying the presence of Leishmania spp. and the blood source of the collected female sand flies. Part of the females were obtained from the Parque Estadual do Rio Doce, and part was collected using 19 ligth traps distributed in residences of Timóteo. For molecular studies of Leishmania spp. DNA, the ITS1 gene was used, and in the search for blood source, the CytB gene was used and positive samples were sequenced. The study demonstrated that there are at least three species of Leishmania circulating in the study areas: Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (V.) guyanensis. Nyssomyia whitmani was the predominant sand fly species in the urban area of Timóteo with a positive diagnosis for the presence of Leishmania braziliensis DNA. We found the presence of blood from Gallus gallus (Chicken) and Sus scrofa (Pig) in sand flies. The present study demonstrates that Leishmania braziliensis is the main agent of cutaneous leishmaniasis in the study area, with the effective participation of Nyssomyia whitmani as the vector and both Gallus gallus and Sus scrofa acting as a food source for female sand flies, and helping maintaining the sand fly life.

Rhabdoviral Endogenous Sequences Identified in the Leishmaniasis Vector Lutzomyia longipalpis Are Widespread in Sandflies from South America.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

DNA Barcoding Culicoides Biting Midges (Diptera: Ceratopogonidae) in Northeast Brazil.

Biting midges of the genus Culicoides are small insects associated with the transmission of several pathogens, which requires the correct identification of the species, for implementation of effective strategies against these insects. However, many species are difficult to identify only by morphological characters. Therefore, the use of molecular methods can help in the taxonomy and systematics of this group. Here, the DNA barcode approach was evaluated for nine species of Culicoides from the State of Maranhão, Brazil. We generated 39 sequences from a 476 bp (base pairs) fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene. To assess the usefulness of COI barcodes for the identification of these species, paired genetic distances from intra and interspecific comparisons and phylogenetic trees were generated in MEGA and RAxML/BEAST softwares, respectively. In addition, species delimitation was performed using the PTP, GMYC, and ABGD algorithms. The intra and interspecific genetic distances showed a clear distinction between them, demonstrating that, for the taxa studied, there can hardly be ambiguous identifications with barcodes. In the same sense, the phylogenetic reconstruction resulted in well-supported clades for all morphospecies analyzed.

DNA barcoding of sand flies (Diptera, Psychodidae, Phlebotominae) from the western Brazilian Amazon.

The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.

Effects of anti-Leishmania compounds in the behavior of the sand fly vector Lutzomyia longipalpis.

BACKGROUND: Leishmaniasis is an infectious parasitic disease caused by pathogens of the genus Leishmania transmitted through the bite of adult female sand flies. To reduce case numbers, it is necessary to combine different control approaches, especially those aimed at the sand fly vectors. Innovative forms of control with the use of attractive sugar baits explored the fact that adult sand flies need to feed on sugars of plant origin. Leishmania parasites develop in the gut of sand flies, interacting with the sugars in the diet of adults. Recent studies have shown that sugar baits containing plant-derived compounds can reduce sand fly survival, the number of parasites per gut, and the percentage of infected sand flies. Several synthetic compounds produced from naphthoquinones and pterocarpans have anti-parasitic activity on Leishmania amazonensis and/or Leishmania infantum in cell culture. This work aimed to assess the inclusion of these compounds in sugar baits for blocking transmission, targeting the development of the Leishmania parasite inside the sand fly vector. RESULTS: We evaluated the attractant or repellent properties of these compounds, as well as of the reference compound N,N'-diethyl-m-toluamide (DEET), in sugar baits. We also observed changes in feeding preference caused by these compounds, looking for anti-feeding or stimulation of ingestion. Pterocarpanquinone L4 and pentamidine showed attractant and repellent properties, respectively. CONCLUSION: Based on the effects in feeding preference and intake volume, pterocarpanquinone L6, and the pyrazole-derived compound P8 were chosen as the most promising compounds for the future development of anti-Leishmania sugar baits. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Diversity of Culicoides (Diptera: Ceratopogonidae) in Rural Settlements and Degraded Seasonal Deciduous Forests.

Biting midges are widespread in Brazilian natural ecosystems. However, deforestation and other activities that impact the environment are reducing natural habitats where biting midges proliferate. The objective of this study was to verify whether there is variation in the composition, richness, abundance, and seasonality of biting midges between wild and rural environments, in a forest area with intense deforestation. Biting midges were captured using 6 traps installed at an average height of 1.5 m in the peridomicile, intradomicile, and deciduous seasonal forests, once a month from May 2012 to April 2013. In total, 2,182 specimens of 13 species of the genus Culicoides were captured. Species richness was similar in the intradomicile (13 species), forest (12), and peridomicile (11), but species diversity was greater in the peridomicile (H' = 0.803) compared with the intradomicile (H' = 0.717) and forest (H' = 0.687). The order of species dominance varied between the forest (Culicoides paucienfuscatus Barbosa > Culicoides leopodoi Ortiz > Culicoides foxi Ortiz > Culicoides ignacioi Forattini) and peridomicile + intradomicile habitats (C. paucienfuscatus > C. foxi > C. filariferus Hoffman > C. ignacioi). The activity of these dipterans was strongly influenced by meteorological variables, as biting midges are predominant in the rainy season (80.7% of specimens), when higher rainfall, relative humidity, and lower temperatures prevail. The abundance of biting midges was higher in the peridomicile + intradomicile (83.7% of specimens) compared with the degraded forest (16.3%), a result that reflects the loss of forest habitat due to intense and progressive deforestation.

New species of Boreofairchildia Wagner amp; Stuckenberg and Laurenceomyia Wagner amp; Stuckenberg (Diptera: Psychodidae: Bruchomyiinae) from Brazil.

Two new species of Bruchomyiinae Macquart from Brazil are described and illustrated. Laurenceomyia peixotoi Santos, Brazil Pinto sp. nov. to Amazon Forest and Boreofairchildia alexanderi Santos, Brazil Pinto sp. nov. to Atlantic Forest.

Diagnosis and identification of Leishmania species in patients with cutaneous leishmaniasis in the state of Roraima, Brazil's Amazon Region.

BACKGROUND: Cutaneous leishmaniasis (CL) is an endemic disease in Brazil that is highly prevalent in the northern region of the country. Although there is a continuous and growing number of cases registered in the state of Roraima, there is limited information regarding the species of Leishmania that affect the human population. In this study, we aimed to characterize which Leishmania species cause human disease in those presenting with cutaneous leishmaniasis in endemic areas of the State of Roraima. METHODS: We conducted a prospective surveillance study between 2016 to 2018 in health centers located in the State of Roraima, Brazil. Participants with clinical suspicion of CL were enrolled and provided lesion samples for parasitological detection by microscopy. A subset of the samples was tested by polymerase chain reaction and sequencing of the internal transcribed spacer 1 (ITS-1 PCR) for molecular species identification. RESULTS: A total of 262 participants were enrolled in this study. Of those, 129 (49.27%) were positive by parasitological examination. Most positive subjects (81.58%) were male, and most cases presented a single lesion (80.26%). ITS-1 PCR and sequencing on a subset of 76 samples allowed us to detect nine different species of Leishmania: L. (V.) braziliensis, L (V.) panamensis, L. (V.) guyanensis, L. (V.) naiffi, L. (V.) shawi, L.(V.) utingensis, L. (V.) lindenbergi, L. (L.) amazonensis and L. (L.) mexicana. CONCLUSIONS: Our study provides the first assessment of circulating species of Leishmania in the State of Roraima, Brazil, and shows the high diversity in this region. This study opens the path for further research on the transmission of leishmaniasis in the northernmost Brazilian State including vector and reservoir surveillance as well as for intensification of investigation and control activities against CL in the region.

Parasitological and molecular diagnosis of cutaneous leishmaniasis among indigenous peoples in the state of Roraima, Brazil.

INTRODUCTION: We diagnose cases of cutaneous leishmaniasis (CL) among indigenous peoples of the state of Roraima, Brazil, and discuss some aspects of its epidemiology. METHODS: Skin imprints, and lesion exudate samples collected on filter paper were examined using parasitological and molecular techniques, respectively. RESULTS: Of 30 indigenous individuals, representing several ethnic groups, with suspected cases of CL, 27 (90%) tested positive for Leishmania spp. by PCR, and 21 (70%) by parasitological microscopy. CONCLUSIONS: Cutaneous leishmaniasis is indistinctly present among indigenous peoples from different regions of the state of Roraima. Individuals from seven of the ten existing ethnic groups in the state tested positive for CL, demonstrating the need for further investigation of the disease among these ethnic groups.

Parasitological and molecular diagnosis of cutaneous leishmaniasis among indigenous peoples in the state of Roraima, Brazil

Abstract INTRODUCTION: We diagnose cases of cutaneous leishmaniasis (CL) among indigenous peoples of the state of Roraima, Brazil, and discuss some aspects of its epidemiology. METHODS: Skin imprints, and lesion exudate samples collected on filter paper were examined using parasitological and molecular techniques, respectively. RESULTS: Of 30 indigenous individuals, representing several ethnic groups, with suspected cases of CL, 27 (90%) tested positive for Leishmania spp. by PCR, and 21 (70%) by parasitological microscopy. CONCLUSIONS: Cutaneous leishmaniasis is indistinctly present among indigenous peoples from different regions of the state of Roraima. Individuals from seven of the ten existing ethnic groups in the state tested positive for CL, demonstrating the need for further investigation of the disease among these ethnic groups.

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil.

Leishmaniasis encompasses a group of diverse clinical diseases caused by protozoan parasites of the Leishmania genus. This disease is a major public health problem in the New World affecting people exposed in endemic regions. The city of Governador Valadares (Minas Gerais/Brazil) is a re-emerging area for visceral leishmaniasis, with 191 human cases reported from 2008 to 2017 and a lethality rate of 14.7%. The transmission of the parasite occurs intensely in this region with up to 22% of domestic dogs with positive serology for the visceral form. Lu. longipalpis is one of the most abundant sand fly species in this area. Despite this scenario, so far there is no information regarding the circulating Leishmania species in the insect vector Lutzomyia longipalpis in this focus. We collected 616 female Lutzomyia longipalpis sand flies between January and September 2015 in the Vila Parque Ibituruna neighborhood (Governador Valadares/MG), which is located on a transitional area between the sylvatic and urban environments with residences built near a preserved area. After DNA extraction of individual sand flies, the natural Leishmania infections in Lu. longipalpis were detected by conventional PCR, using primers derived from kDNA sequences, specific for L. (Leishmania) or L. (Viannia) subgenus. The sensitivity of these PCR reactions was 0.1 pg of DNA for each Leishmania subgenus and the total infection rate of 16.2% (100 positive specimens). Species-specific PCR detected the presence of multiple Leishmania species in infected Lu. longipalpis specimens in Governador Valadares, including L. amazonensis (n = 3), L. infantum (n = 28), L. (Viannia) spp. (n = 20), coinfections with L. infantum and L. (Viannia) spp. (n = 5), and L. (Leishmania) spp (n = 44). Our results demonstrate that multiple Leishmania species circulate in Lu. longipalpis in Governador Valadares and reveal a potential increasing risk of transmission of the different circulating parasite species. This information reinforces the need for epidemiological and entomological surveillance in this endemic focus, and the development of effective control strategies against leishmaniasis.

Transmission blocking sugar baits for the control of Leishmania development inside sand flies using environmentally friendly beta-glycosides and their aglycones.

BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.

Morphological and phylogenetic analyses of Lutzomyia migonei from three Brazilian states.

Lutzomyia migonei is incriminated as a vector of Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. Recently, this phlebotomine sand fly species has been suggested as a vector for Leishmania infantum, which causes zoonotic visceral leishmaniasis. Considering the widespread distribution of Lu. migonei in South America, the existence of isolated populations has been hypothesized. Three Lu. migonei populations, two from north-eastern Brazil (Machados, Pernambuco State, and Baturité, Ceará State) and other from the south-eastern region (Niterói, Rio de Janeiro State) were analysed both morphologically and genetically. Though no significant morphological differences were found amongst the sand fly specimens analysed, discriminant analysis based on specific morphometric characters (i.e., length of wing, antennal segment 3 and coxite for males, and length of wing and antennal segment 3 for females), showed that specimens from Machados were closer to Baturité than to Niterói. The molecular analysis of cytochrome c oxidase subunit I gene sequences also supported this observation by the distinct separation of two monophyletic clades, grouping specimens from Machados and Baturité separately from those of Niterói. Our results suggest the existence of different populations within the distribution range of Lu. migonei. Whether these populations are reproductively isolated and/or present differences in terms of vector competence/capacity for L. braziliensis and L. infantum needs to be further investigated.

Molecular diagnosis of cutaneous leishmaniasis in an endemic area of Acre State in the Amazonian Region of Brazil.

INTRODUCTION: This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS: Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS: Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS: These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.

Molecular diagnosis of cutaneous leishmaniasis in an endemic area of Acre State in the Amazonian Region of Brazil

Abstract INTRODUCTION This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.

Ecology, feeding and natural infection by Leishmania spp. of phlebotomine sand flies in an area of high incidence of American tegumentary leishmaniasis in the municipality of Rio Branco, Acre, Brazil.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to their involvement in the zoonotic transmission of Leishmania spp. to vertebrates. The aim of this work was to study the ecology of the sand fly fauna of two types of environments, a rural environment (the Transacreana Road) and an urban park (Horto Florestal Park), both located in the municipality of Rio Branco in the state of Acre, Brazil. Additionally, this study intended to investigate Leishmania infection and blood meal sources of these sand flies using molecular techniques. METHODS: The sand fly fauna was studied in different environments (i.e. forest and peridomestic environments in a rural area, and an urban forest) using Shannon traps and HP light traps to collect sand fly specimens over 13 consecutive months (December 2014 to January 2016). For investigating natural infection by Leishmania and the source of sand fly blood meals, DNA samples were extracted from female sand flies and subjected to polymerase chain reaction targeting ITS1 and cytb genes. DNA sequencing was subsequently used to identify species of Leishmania and the source of blood meals. RESULTS: A total of 2515 individual sand flies of 43 species were collected and identified, Trichophoromyia auraensis (839; 33.35%), Trichophoromyia spp. (537; 21.35%) and Evandromyia saulensis (187; 7.43%) were more abundant in the rural area (S = 41 species) than in the urban forest. No significant differences were found in species richness between forest and peridomestic environments in the rural area (H = 0.04; P > 0.05), but a larger number of species was found in the forest. Leishmania DNA was sequenced in 13 samples, confirming the presence of L. (V.) braziliensis in Th. auraensis (n = 1), Ev. saulensis (n = 2), Ev. walkeri (n = 1), Ps. llanosmartinsi (n = 1), Pi. nevesi (n = 2), Ps. davisi (n = 1), Ps. ayrozai (n = 1), Pa. aragaoi (n = 1), Ny. antunesi (n = 1) and Ev. infraspinosa (n = 1). Only Ps. ayrozai possessed a sequence similar to that of L. (V.) guyanensis (99%). Through microscopic analysis, five specimens of Ev. saulensis were found to possess flagellate forms in the hindgut, with an infection rate of 2.4%. Samples from 33 fed females were submitted to cytb gene amplification, for which sequencing determined that all were similar to the sequence deposited on GenBank for Gallus gallus (domestic chicken). CONCLUSIONS: The high abundance of Trichophoromyia auraensis and Ev. saulensis, and the detection of L. (V.) braziliensis DNA, suggests that both species may be vectors of American tegumentary leishmaniasis. Psychodopygus ayrozai was found to be infected by L. (V) braziliesnsis and L. (V.) guyanensis, and although collected in low abundance, it may be a potential vector in the region. The sand fly fauna was found to be rich and diverse with predominance of the genus Psychodopygus. Identification of food sources of fed females showed that 100% amplified a gene region compatible with the domestic chicken, which although considered refractory in the disease transmission cycle, may have an influence on the population dynamics of sand flies.

Trichophoromyia auraensis is a putative vector

The sandfly Trichophoromyia auraensis has recently evolved as a proven vector of Leishmania (Viannia) endemic to state of Acre in the north of Brazil. This note is intended to propose a correction in the report of the first occurrence of natural infection of Leishmania (Viannia) in this species. We and the other scientific groups reinforced that Tr. auraensis is a possible vector involved in the transmission of American cutaneous leishmaniasis in Acre, Brazil.

First description of Leishmania (Viannia) infection in Evandromyia saulensis, Pressatia sp. and Trichophoromyia auraensis (Psychodidae: Phlebotominae) in a transmission area of cutaneous leishmaniasis in Acre state, Amazon Basin, Brazil.

Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.

First description of Leishmania (Viannia) infection in Evandromyia saulensis, Pressatia sp. and Trichophoromyia auraensis (Psychodidae: Phlebotominae) in a transmission area of cutaneous leishmaniasis in Acre state, Amazon Basin, Brazil

Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.