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

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.

Field randomized trial to evaluate the efficacy of the Leish-Tec® vaccine against canine visceral leishmaniasis in an endemic area of Brazil.

BACKGROUND: A canine vaccine remains a promising approach for effective control of visceral leishmaniasis (VL), given its complex epidemiology in areas where zoonotic VL is prevalent. Leish-Tec(®) is a recombinant vaccine, based on the Leishmania A2 antigen, against canine VL (CVL). It is, since 2014, the single commercial vaccine licensed in Brazil. Here, Leish-Tec(®) efficacy was estimated through a randomized field trial (RFT), in a highly VL endemic area. METHODS: The RFT was conducted from 2008 to 2010 in an endemic area of southeastern Brazil, presenting a CVL seroprevalence of 41.9%. Eight hundred forty-seven seronegative dogs were randomly selected to receive Leish-Tec(®) (n=429) or placebo (n=418). Animals were followed up by clinical, serological, and parasitological exams for 18 months. The CVL incidence in both groups was compared through proportion analysis. RESULTS: A significant reduction in the number of cases of CVL was observed in the vaccine group, as compared with the placebo group, whether efficacy was estimated according to parasitological results (71.4%; 95% CI: 34.9-87.3%; p=0.001; risk ratio=0.287), by adding results of xenodiagnosis and parasitological exams (58.1%; 95% CI: 26.0-76.3%; p=0.002; risk ratio=0.419). Among the animals that converted to a positive anti-A2 serology, efficacy reached 80.8% (95% CI: 37.6-94.1%, p=0.001; risk ratio=0.192). Xenodiagnosis has detected a reduction of 46.6% (p=0.05) in transmission to sand flies from vaccinated animals presenting anti-A2 positive serology. CONCLUSION: The Leish-Tec(®) vaccine proved significantly effective for prophylaxis of CVL, after natural challenge assured by transmission of Leishmania parasites, in a highly endemic area. Noteworthy, this report has unveiled the complexity of performing a RFT for anti-CVL vaccines in Brazil, which may be helpful for designing of future studies.

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.

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. .

Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil.

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.