Ana Cristina Vianna Mariano da Rocha Lima

Naturally Infected Lutzomyia Sand Flies in a Leishmania-Endemic Area of Brazil

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.

Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

ABSTRACT Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (=Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (=Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis.

The vectorial capacity of Rhipicephalus sanguineus in the transmission of canine visceral leishmaniasis has been evaluated through a laboratory-controlled experiment. One healthy Leishmania-free dog and two dogs naturally infected with Leishmania were infested with R. sanguineus in various stages of development. Engorged larvae, unfed nymphs, engorged nymphs, unfed adults, engorged female adults and fed male adults were collected from the experimental animals and examined for Leishmania infection by optical microscopy, polymerase chain reaction (PCR) and parasite culture. Leishmania forms were not detected in any of the 433 smears prepared from engorged colonies nor in any of the 118 smears prepared from unfed colonies. However, one flagellate structure was identified in one of the smears. All pools of R. sanguineus that had fed on the infected dogs tested PCR-positive for Leishmania DNA, with the single exception of the pool of engorged larvae. In contrast, all pools of ticks that had fed on the Leishmania-free dog were PCR-negative. Leishmania growth was not observed in any of the tick colonies following incubation on culture medium. Considering that no Leishmania forms were identified in any of the meticulously analysed smears derived from engorged colonies of R. sanguineus, it appears somewhat unlikely that the maintenance and multiplication of Leishmania occurs within the tick.

Phlebotomine Sand Fly Fauna and Leishmania Infection in the Vicinity of the Serra do Cipó National Park, a Natural Brazilian Heritage Site

In the New World, the leishmaniases are primarily transmitted to humans through the bites of Leishmania-infected Lutzomyia (Diptera: Psychodidae) phlebotomine sand flies. Any or both of two basic clinical forms of these diseases are endemic to several cities in Brazil—the American cutaneous leishmaniasis (ACL) and the American visceral leishmaniasis (AVL). The present study was conducted in the urban area of a small-sized Brazilian municipality (Jaboticatubas), in which three cases of AVL and nine of ACL have been reported in the last five years. Jaboticatubas is an important tourism hub, as it includes a major part of the Serra do Cipó National Park. Currently, no local data is available on the entomological fauna or circulating Leishmania. During the one-year period of this study, we captured 3,104 phlebotomine sand flies belonging to sixteen Lutzomyia species. In addition to identifying incriminated or suspected vectors of ACL with DNA of the etiological agent of AVL and vice versa, we also detected Leishmania DNA in unexpected Lutzomyia species. The expressive presence of vectors and natural Leishmania infection indicates favorable conditions for the spreading of leishmaniases in the vicinity of the Serra do Cipó National Park.

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.

Epidemiological aspects of vector; parasite; and domestic reservoir in areas of recent transmission and no reported human cases of visceral leishmaniasis in Brazil.

About 97% of the human cases of the American visceral leishmaniasis (VL) occur in Brazil. In the last few years, the disease expanded to medium- and large-sized cities, in which surveillance and control actions have been intensified, in an effort to control VL spreading. Our two-year study was conducted in Belo Horizonte, the sixth most populous city in Brazil, which is endemic for VL. We focused in two particular districts of recent transmission of the disease, with no reported human cases and submitted to minor surveillance and control actions. Our aim was to draw an epidemiological profile of the local situation concerning Lutzomyia vector, Leishmania parasites, and the main domestic reservoirs (dogs). Lutzomyia longipalpis comprised 96.5% of the total phlebotomine sand flies captured and displayed an expressive minimal infection rate by Leishmania infantum (16.7%). Positive correlations were found between the population densities of L. longipalpis, rainfall and temperature. L. infantum was also detected in the cortelezzii complex and, for the first time, in Lutzomyia lloydi. Leishmania braziliensis, an etiological agent of the American cutaneous leishmaniasis, was also identified in L. longipalpis. Among the 1408 dogs serologically tested by standard enzyme-linked and fluorescence immune assays (ELISA/IFA) 3.6% were positive for VL. L. infantum DNA and Leishmania parasites were identified in 100% and 72.5% of the seropositive dogs, respectively. The co-positivity of other diagnostic tests for VL-Leishmania-nested PCR, imprint and myeloculture-was compared to the standard serology. Both symptomatic or asymptomatic dogs displayed an equal average number of positive diagnostic tests for VL. The districts studied display favorable conditions for the rapid spreading of human infection, in terms of L. longipalpis population density, and presence of L. infantum in both vector and main reservoir.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

INTRODUCTION Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. METHODS We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. RESULTS The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. CONCLUSIONS The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Reliability of techniques used in the diagnosis of canine visceral leishmaniasis by the national control program in Brazil: A survey in an area of recent transmission

One of the key components of the Brazilian Program for the Control of Visceral Leishmaniasis (PCLV) is the euthanasia of Leishmania-infected canine reservoirs, the detection of which depends on a screening procedure involving a Dual Path Platform® (DPP) immunoassay and a confirmatory enzyme-linked immunosorbent assay (ELISA). The aims of the present study were to evaluate the reliability of these techniques in a region of recent transmission of canine VL, to follow up the seroconversion 3-4 months after the initial diagnosis of DPP reactive but ELISA indeterminate or non-reactive dogs, and to identify the species of Leishmania in circulation in the area. Each animal was submitted to DPP under field conditions, performed by municipal health workers using peripheral blood (DPP-field), to DPP under laboratory conditions using serum (DPP-lab) and to ELISA using serum. The agreements between the tests were determined using McNemars χ2 test, Cohens kappa coefficient (k) at the 95% confidence interval and prevalence-adjusted bias-adjusted kappa (PABAK). Of the 1130 dogs examined, 74.2% were non-reactive in all three tests applied. Based on the PCLV positive-infection criterion, seroprevalence was 8.9% (101/1130) with 83.2% (84/101) of infected animals showing reactivity in all three tests while 7.8% (8/101) were reactive in DPP-field and ELISA and 8.9% (9/101) in DPP-lab and ELISA. The proportions of disagreements were substantial in all comparisons. Inter-rater reliability between DPP-field and ELISA (k=0.55; PABAK=0.78) and DPP-lab and ELISA (k=0.59; PABAK=0.81) were considered moderate, while that between DPP-field and DPP-lab (k=0.61; PABAK=0.79) was classified as marginally good. The proportion of seroconversions in DPP reactive animals that were initially ELISA indeterminate was significantly higher than in those that were DPP reactive but initially ELISA non-reactive. Restriction fragment length polymorphism analysis revealed the presence of Leishmania infantum, the etiologic agent of VL, in bone marrow samples from VL-infected animals. Our data showed that the techniques and protocols currently employed in the PCLV screening approach are not entirely reliable. Further consideration should be given to monitoring dogs with undetermined results in ELISA and a better training should be provided for health workers responsible for performing DPP tests applied under field conditions.

Seasonality of sand flies (Diptera: Psychodidae) and Leishmania DNA detection in vector species in an area with endemic visceral leishmaniasis

BACKGROUND Leishmaniases are a serious health problem in southeast Brazil, including the city of Belo Horizonte (BH), Minas Gerais state (MG), where there are high rates of incidence and mortality due to visceral leishmaniases. BH is divided into nine sanitary districts (SD) of which one, the Venda Nova SD, was selected for this study because it has high rates of positivity for canine leishmaniasis and high incidence of human leishmaniasis. OBJECTIVES This study aimed to survey the sand fly fauna in Venda Nova SD from August 2011 to July 2013 and perform a descriptive analysis of the vector population. METHODS The sampling was carried out using automatic HP light traps at all covered areas of the Venda Nova SD, in a total of eighteen light traps. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A simple environmental description was done for it area and Kernel estimation was used to infer vector density for each study site. FINDINGS A total of 2,427 sand fly specimens belonging to eight species and five genera were collected of which 95.3% were Lutzomyia longipalpis. The seasonal variation curve was delineated by this species. Lu. longipalpis was the most abundant at all collection points and in all months of the study, and exhibited a natural infection rate of 1.01% for Leishmania infantum and 1.77% for Leishmania braziliensis. MAIN CONCLUSIONS The results show the presence and adaptation of Lu. longipalpis to the anthropic environment of BH and reinforces its role as the main vector of L. infantum in the region.

Seroprevalence and molecular characterization of Leishmania in dogs from an endemic area of zoonotic visceral leishmaniasis in Brazil

Abstract Visceral leishmaniasis (VL) can cause large-scale and tenacious epidemics with high fatality rates. Current seroprevalence and circulating Leishmania species were evaluated in dogs domiciled in the municipality of Sabará, a small historic and touristic city in the Brazilian state of Minas Gerais. A total of 3926 dogs domiciled in seven different districts of Sabará were serologically tested for canine visceral leishmaniasis (CVL) by indirect enzyme-linked immunosorbent (ELISA) and immunofluorescence (IFA) assays, in a two-years census survey (2011–2012). The average positivity rate of canine infection was 3.4%. Three additional diagnostic tests – imprint/smear direct parasitological, molecular (LnPCR) and myeloculture – were performed in a random sample of fifty seropositive dogs composed of symptomatic (39) and asymptomatic (eleven) animals. LnPCR showed 100% of positivity for Leishmania DNA in, at least, one among four tissue samples tested (mesenteric lymph node, skin, spleen and bone marrow), independently of the clinical canine group. Higher and statistically equivalent positivity rates (98% and 96%) for Leishmania DNA were found in canine lymph node and spleen. Asymptomatic dogs showed expressive positivity rates in all three additional diagnostic techniques. Leishmania infantum was confirmed as the etiological agent of CVL in Sabará.