Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Dogs with canine visceral leishmaniasis have a boost of extracellular vesicles and miR-21-5p up-expression.

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 1010 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 1010 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Canine serological survey and dog culling ant its relationship with human visceral leishmaniasis in an endemic urban area.

BACKGROUND: Visceral leishmaniasis is an important but neglected disease that is spreading and is highly lethal when left untreated. This study sought to measure the Leishmania infantum seroprevalence in dogs, the coverage of its control activities (identification of the canine reservoir by serological survey, dog culling and insecticide spraying) and to evaluate its relationship with the occurrence of the disease in humans in the municipalities of Araçatuba and Birigui, state of São Paulo, Brazil. METHODS: Information from 2006 to 2015 was georeferenced for each municipality and modeling was performed for the two municipalities together. To do this, latent Gaussian Bayesian models with the incorporation of a spatio-temporal structure and Poisson distribution were used. The Besag-York-Mollie models were applied for random spatial effects, as also were autoregressive models of order 1 for random temporal effects. The modeling was performed using the INLA (Integrated Nested Laplace Approximations) deterministic approach, considering both the numbers of cases as well as the coverage paired year by year and lagged at one and two years. RESULTS: Control activity coverage was observed to be generally low. The behavior of the temporal tendency in the human disease presented distinct patterns in the two municipalities, however, in both the tendency was to decline. The canine serological survey presented as a protective factor only in the two-year lag model. CONCLUSIONS: The canine serological coverage, even at low intensity, carried out jointly with the culling of the positive dogs, suggested a decreasing effect on the occurrence of the disease in humans, whose effects would be seen two years after it was carried out.

Canine visceral leishmaniasis in Araçatuba, state of São Paulo, Brazil, and its relationship with characteristics of dogs and their owners: a cross-sectional and spatial analysis using a geostatistical approach.

BACKGROUND: The incidence of visceral leishmaniasis (VL), one of the most important neglected diseases worldwide, is increasing in Brazil. The objectives of this study were to determine the canine VL (CanL) seroprevalence in an urban area of Araçatuba municipality and to evaluate its relationship with the characteristics of dogs and their owners. RESULTS: The CanL seroprevalence in the study area was 0.081 (95% credible interval [CI]: 0.068-0.096). The following covariates/categories were positively associated with the occurrence of a seropositive dog: more than 10 dogs that had lived in the house (odds ratio [OR] = 2.36; 95% CI: 1.03-5.43) (baseline: 0-10 dogs); house with dogs that previously died of VL (OR = 4.85; 95% CI: 2.65-8.86) or died of causes other than old age (OR = 2.26; 95% CI: 1.12-4.46) (baseline: natural or no deaths); dogs that spent the day in a sheltered backyard (OR = 2.14; 95% CI: 1.05-4.40); dogs that spent the day in an unsheltered backyard or the street (OR = 2.67; 95% CI: 1.28-5.57) (baseline: inside home). Spatial dependence among observations occurred within about 45.7 m. CONCLUSIONS: The number of dogs that had lived in the house, previous deaths by VL or other cause, and the place the dog stayed during the day were associated with the occurrence of a VL seropositive dog. The short-distance spatial dependence could be related to the vector characteristics, producing a local neighbourhood VL transmission pattern. The geostatistical approach in a Bayesian context using integrated nested Laplace approximation (INLA) allowed to identify the covariates associated with VL, including its spatially dependent transmission pattern.

The shadows of a ghost: a survey of canine leishmaniasis in Presidente Prudente and its spatial dispersion in the western region of São Paulo state, an emerging focus of visceral leishmaniasis in Brazil.

BACKGROUND: Visceral leishmaniasis is an emerging zoonosis and its geographic distribution is restricted to tropical and temperate regions. Most of the individuals infected in Latin America are in Brazil. Despite the control measures that have been adopted, the disease is spreading throughout new regions of the country. Domestic dogs are involved in the transmission cycle and are considered to be the main epidemiologic reservoir of Leishmania infantum (syn. L. chagasi). Our aim was to determine the prevalence of canine leishmaniasis (CL) and Ehrlichiosis infection in Presidente Prudente as well as the spatial dispersion of the disease in the western region of São Paulo state. METHODS: Dogs underwent clinical examination and symptoms related to CL were recorded. Anti- Leishmania antibodies were detected using ELISA, rK39-immunocromatographic tests (DPP), and an indirect fluorescent antibody test (IFAT). Anti-E. canis antibodies were detected by IFAT. A follow-up was conducted in dogs that were positive in the ELISA at the baseline study. Data on the spatial distribution of L. longipalpis and CL in São Paulo state were obtained from Brazilian public health agencies. RESULTS: Serum samples from 4547 dogs were analyzed. The seroprevalence of CL was 11.2% by ELISA and 4.5 % by IFAT. In the follow-up, seroprevalence was 32.9% by ELISA, 15.3% by IFAT, 11.8 % by DPP test, and 66.5% for E. canis. There was a significant positive association between Leishmania and E. canis infection (P < 0.0001). In the follow-up, clinical examinations revealed symptoms compatible with CL in 33.5% of the dogs. L. longipalpis was found in 24 and CL in 15 counties of the Presidente Prudente mesoregion. The dispersion route followed the west frontier of São Paulo state toward Paraná state. CONCLUSIONS: Low CL and high ehrlichiosis prevalence rates were found in Presidente Prudente city. This emerging focus of CL is moving through the western region of São Paulo state toward the border of Paraná state. Integrated actions to fight the vector, parasites, infected dogs, and humans are needed to monitor the disease and implement strategies for epidemiologic control.

Spatial epidemiology of American cutaneous leishmaniasis in a municipality of west São Paulo State, Brazil.

BACKGROUND & OBJECTIVES: In the last decade, in the state of São Paulo, 5898 cases of cutaneous leishmaniasis (CL) were reported. This study was undertaken to analyze the epidemiology of CL in the municipality of Teodoro Sampaio, in São Paulo State, Brazil, based on a geographic approach, as very little is known of the relationship between CL and the spatial transformation process. METHODS: This is a population-based quantitative, descriptive and cross-sectional case study. Surveys of the official notifications in the healthcare center and official sources from 1998 to 2011 were analysed. The data were described based on statistics and the Kernel method to detect hotspots of transmission. RESULTS: The age group between 21 and 40 yr was most affected, with 24 cases (57.9%). Of the 41 cases reported between 1998 and 2011, 33 cases were having low education status and 31 cases (75.6%) were males. The spatial and temporal distribution was aggregated in three-year periods which permitted the identification of two microfoci, in periods I (1998-2000) and III (2005-2007). INTERPRETATION & CONCLUSION: The disease has presented, in recent years, a pattern of sporadic transmission or microfoci, and continues to maintain enzootic cycles of Leishmania in a sylvatic environment, ensuring the perpetuation of the pathogen in nature, and the risk of emergence of new cases of CL in domestic animals and humans.

Extracellular Vesicles from Leishmania (Leishmania) infantum Contribute in Stimulating Immune Response and Immunosuppression in Hosts with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host-EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts.

Genetic variability of Leishmania (Leishmania) infantum causing human visceral leishmaniasis in the Southeastern Brazil.

Leishmania infantum is a protozoan that causes visceral leishmaniasis (VL) in the Americas and some regions of Europe. The disease is mainly characterized by hepatosplenomegaly and fever, and can be fatal. Factors related to the host and parasite can contribute to the transmission of Leishmania and the clinical outcome. The intraspecific genetic variability of L. infantum strains may be one of these factors. In this study, we evaluated the genetic variability of L. infantum obtained from bone marrow smear slides from patients in the Sao Paulo State, Brazil. For this, the minicircle of the kDNA hypervariable region was used as target by Sanger sequencing. By analyzing the similarity of the nucleotides and the maximum likelihood tree (Fasttree), we observed a high similarity (98%) among samples. Moreover, we identified four different profiles of L. infantum. In conclusion, L. infantum strains from Sao Paulo State, Brazil, showed low diversity measured by minicircle of the kDNA hypervariable region.

The path of canine visceral leishmaniasis versus the path of Center for Zoonoses Control: contributions of spatial analysis to health.

Visceral leishmaniasis (VL) is a public health problem in Brazilian municipalities. As much as there is a planning of public policies regards VL in São Paulo State, new cases have been reported and spread. This paper aims to discuss how the Center for Zoonoses Control conducts its actions spatially in endemic city of Presidente Prudente, São Paulo State. Data are from the Municipal Health Department of Presidente Prudente, Adolfo Lutz Institute, and Brazilian Institute of Geography and Statistics. We spatially estimated the dog population per census tract and used geoprocessing tools to perform choropleth maps, spatial trends, and spatial autocorrelation. We found a spatial pattern of higher prevalence in the city's outskirt and a positive statistically significant spatial autocorrelation (I = 0.2, p-value < 0.000) with clusters of high-high relationships in the Northwest part of the city. Moreover, we identified a different direction in the path of the conducted serosurveys versus the canine VL trend, which stresses the fragility of the Center for Zoonoses Control actions to control the disease. The Center for Zoonoses Control always seems to chase the disease. The spatial analysis may be useful for rethinking how the service works and helps in public policies.

Efficacies of insecticide dog collars against visceral leishmaniasis in low and high-income areas and the effects for non-collared neighbor dogs.

Previous studies demonstrated that insecticide collars are highly effective in reducing canine visceral leishmaniasis (CVL); however, it is unclear if the efficacy differs by socioeconomic conditions across diverse communities. This study aimed fourfold: (i) to evaluate the protection of 4% impregnated deltamethrin collared (DMC) dogs in different areas of an endemic city for visceral leishmaniasis (VL); (ii) to analyze socioeconomic variables with the seroconversion rates; (iii) to analyze the indirect effect of DMC on untreated dogs in areas of intervention; and, (iv) to evaluate the potential transmission to other dogs in the same household when one positive dog is present. The study employed the municipality of Bauru, São Paulo, Brazil, as the area of interest and used Geographic Information System tools to fit binary logistic regression models.  Dogs were divided into three cohort studies: intervention with DMC (I), indirect effect of DMC (IE), and control (C). Pre-intervention, lower mean income was associated with higher rates of CVL and a 142% increase in the odds of transmission (OR = 1.42, p-value = 0.001, CI 1.14,1.77). Post-intervention, lower-income areas depicted greater efficacy (76%) than higher-income areas (45%). The overall efficacy of DMC in preventing CVL was 63%; however, seroconversion rates were higher for IE (6.02%) than C (3.78%), revealing the failure of the indirect protection of DMC to manage the spread of the disease among the general non-wearing DMC canine population living in the same area. The protected dogs may repel the vectors, and non-protected dogs attract them, creating a higher transmission rate for non-protected dogs. Greater seroconversion was observed for living with an infected dog (10.20% in IE and 8.75% in C) than for the indirect effect of DMC, demonstrating the social burden of CVL. Overall, uncollared dogs have three times higher odds of being infected with CVL than DMC dogs (p < 0.005), and uncollared dogs living with (an) infected dog(s) in the same household can reach 3.5 times higher odds than those living with negative ones (p < 0.005). The results may assist in enhancing public policies and minimizing inequality in low and middle-income countries that suffer from neglected diseases such as VL.

Impact of the dog population and household environment for the maintenance of natural foci of Leishmania infantum transmission to human and animal hosts in endemic areas for visceral leishmaniasis in Sao Paulo state, Brazil.

When it comes to visceral leishmaniasis (VL) in Brazil, one of the main targets of public health policies of surveillance is the control of domestic canine reservoirs of Leishmania infantum. This paper aims to evaluate the effect of the dog population and household environment for the maintenance of natural foci in the transmission to human and animal hosts in an endemic city for VL, Bauru, in Brazil. We collected 6,578 blood samples of dogs living in 3,916 households from Nov.2019 to Mar.2020 and applied geospatial models to predict the disease risk based on the canine population. We used Kernel density estimation, cluster analysis, geostatistics, and Generalized Additive Models (GAM). To validate our models, we used cross-validation and created a receiver operating characteristic (ROC) curve. We found an overall canine VL (CVL) seroprevalence of 5.6% for the sampled dogs, while for the households, the positivity rate was 8.7%. Odds ratios (OR) for CVL increased progressively according to the number of canines for >2 dogs (OR 2.70); households that already had CVL in the past increased the chances for CVL currently (OR 2.73); and the cases of CVL increase the chances for human VL cases (OR 1.16). Our models were statistically significant and demonstrated a spatial association between canine and human disease cases, mainly in VL foci that remain endemic. Although the Kernel density ratio map had the best performance (AUC = 82), all the models showed high risk in the city's northwest area. Canine population dynamics must be considered in public policies, and geospatial methods may help target priority areas and planning VL surveillance in low and middle-income countries.

[Control of canine visceral leishmaniasis by euthanasia: estimated effect based on a survey and mathematical modeling]. / Controle da leishmaniose visceral canina por eutanásia: estimativa de efeito baseado em inquérito e modelagem matemática.

Visceral leishmaniasis is an emerging and neglected disease that is currently expanding to urban areas. The incidence of human disease is related to canine infection. Araçatuba and Birigui are municipalities (counties) in the state of São Paulo, Brazil, with 8-10% seroprevalence of canine infection and that employ control strategies targeting the canine reservoir, based on serological survey and culling of seropositive dogs. Using data from these control programs to parameterize mathematical models, this study assessed the efficacy of these activities. We estimated that current control is capable of reducing the incidence of canine visceral leishmaniasis (CVL) by approximately 20%. Assuming continuous control and three times the current serological survey activities in Araçatuba and Birigui, culling dogs with a positive CVL diagnosis would be effective for the control of canine infection. Although theoretically possible, in practice the control of CVL with the currently recommended strategies is insufficient, since it would require overcoming the difficulties in these activities, such as lack of material, human, and financial resources, besides associated ethical and legal issues.

A leishmaniose visceral é uma doença emergente e negligenciada em processo de expansão para áreas urbanas. A incidência da doença humana está relacionada com a infecção canina. Araçatuba e Birigui são municípios do Estado de São Paulo, Brasil, com soroprevalência da infecção canina de 8 a 10%, que empregam estratégias de controle voltadas ao reservatório canino baseado em inquérito sorológico e eutanásia dos cães soropositivos. Usando dados desses programas de controle para parametrizar modelos matemáticos, este estudo avaliou a eficácia dessas atividades. Estimamos que o controle atualmente empregado é capaz de reduzir em cerca de 20% a incidência de casos de leishmaniose visceral canina (LVC). Considerando-se um controle contínuo e um esforço das atividades de inquérito sorológico igual ao triplo da média do observado em Araçatuba e Birigui, a atividade de eutanásia de cães com diagnóstico positivo seria efetiva para o controle da infecção canina. Embora teoricamente possível, na prática, o controle da LVC com as estratégias preconizadas atualmente é insuficiente, pois exigiria superpor dificuldades enfrentadas por estas atividades como falta de recursos materiais, humanos e financeiros, além das questões éticas e jurídicas associadas.

La leishmaniasis visceral es una enfermedad emergente y pasada por alto en proceso de expansión hacia áreas urbanas. La incidencia de la enfermedad humana está relacionada con la infección canina. Araçatuba y Birigui son municipios del estado de São Paulo, Brasil, con una seroprevalencia de infección canina de un 8 a un 10% que emplean estrategias de control dirigidas al reservorio canino, basado en una encuesta serológica y eutanasia de los perros seropositivos. Usando datos de esos programas de control para proporcionar parámetros en modelos matemáticos, este estudio evaluó la eficacia de esas actividades. Estimamos que el control actualmente empleado es capaz de reducir cerca de un 20% la incidencia de casos de leishmaniasis visceral canina (LVC). Si se considera un control continuo y un esfuerzo de las actividades de encuesta serológica igual al triple de la media de lo observado en Araçatuba y Birigui, la actividad de eutanasia de perros con diagnóstico positivo sería efectiva para el control de la infección canina. A pesar de que teóricamente es posible, en la práctica el control de la LVC con las estrategias preconizadas actualmente es insuficiente, puesto que exigiría superar dificultades a las que se enfrentan estas actividades como la falta de recursos materiales, humanos y financieros, además de las cuestiones éticas y jurídicas asociadas.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats.

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5 x 10(2) mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Genetic variability of Leishmania (Leishmania) infantum causing human visceral leishmaniasis in the Southeastern Brazil

ABSTRACT Leishmania infantum is a protozoan that causes visceral leishmaniasis (VL) in the Americas and some regions of Europe. The disease is mainly characterized by hepatosplenomegaly and fever, and can be fatal. Factors related to the host and parasite can contribute to the transmission of Leishmania and the clinical outcome. The intraspecific genetic variability of L. infantum strains may be one of these factors. In this study, we evaluated the genetic variability of L. infantum obtained from bone marrow smear slides from patients in the Sao Paulo State, Brazil. For this, the minicircle of the kDNA hypervariable region was used as target by Sanger sequencing. By analyzing the similarity of the nucleotides and the maximum likelihood tree (Fasttree), we observed a high similarity (98%) among samples. Moreover, we identified four different profiles of L. infantum. In conclusion, L. infantum strains from Sao Paulo State, Brazil, showed low diversity measured by minicircle of the kDNA hypervariable region.

Human visceral leishmaniasis and relationship with vector and canine control measures.

OBJECTIVE: Estimate the coverage of control measures of visceral leishmaniasis and relate them with the occurrence of human visceral leishmaniasis in endemic urban area. METHODS: Cases of human and canine visceral leishmaniasis were considered as study population and evaluated by a serological survey conducted in Araçatuba, state São Paulo, from 2007 to 2015. The cases of human visceral leishmaniasis were geocoded by the address of the patients and the canine disease by the address of the dogs' owners. The coverage of serological survey, euthanasia, and insecticide spraying was calculated, as well as the canine seroprevalence and the incidence rates of human visceral leishmaniasis. The relationship between human visceral leishmaniasis and control measures was evaluated, as well as the seroprevalence by comparing maps and by linear regression. The relationship between the canine and the human disease was also evaluated by the Ripley's K function. RESULTS: The incidence rates of human visceral leishmaniasis showed a period of decline (2007 to 2009) and a period of stability (2010 to 2015), a behavior similar to that of canine seroprevalence. In general, the coverage of control measures was low, and the non-association with the incidence of human visceral leishmaniasis can be a result of the period analyzed and of the small number of analyzed units (sectors of the Superintendence for the Control of Endemic Diseases). The distribution of human cases showed spatial dependence with the distribution of seropositive dogs from 2007 to 2009. CONCLUSIONS: This study reaffirmed the relationship between the occurrence of the disease in humans and dogs, it verified a decrease in the rates of visceral leishmaniasis in Araçatuba over time, even at low coverage of control activities. However, further studies are needed to determine if factors beyond monitoring and control measures are involved in the reduction of incidences.

The path of canine visceral leishmaniasis versus the path of Center for Zoonoses Control: contributions of spatial analysis to health / O caminho da leishmaniose visceral canina versus o caminho do Centro de Controle de Zoonoses: contribuições da análise espacial para a saúde / La trayectoria de la leishmaniasis visceral canina versus la trayectoria del Centro para el Control de Zoonoses: contribuciones del análisis espacial a la salud

Visceral leishmaniasis (VL) is a public health problem in Brazilian municipalities. As much as there is a planning of public policies regards VL in São Paulo State, new cases have been reported and spread. This paper aims to discuss how the Center for Zoonoses Control conducts its actions spatially in endemic city of Presidente Prudente, São Paulo State. Data are from the Municipal Health Department of Presidente Prudente, Adolfo Lutz Institute, and Brazilian Institute of Geography and Statistics. We spatially estimated the dog population per census tract and used geoprocessing tools to perform choropleth maps, spatial trends, and spatial autocorrelation. We found a spatial pattern of higher prevalence in the city's outskirt and a positive statistically significant spatial autocorrelation (I = 0.2, p-value < 0.000) with clusters of high-high relationships in the Northwest part of the city. Moreover, we identified a different direction in the path of the conducted serosurveys versus the canine VL trend, which stresses the fragility of the Center for Zoonoses Control actions to control the disease. The Center for Zoonoses Control always seems to chase the disease. The spatial analysis may be useful for rethinking how the service works and helps in public policies.

A leishmaniose visceral (LV) é um problema de saúde pública nas cidades brasileiras. Por mais que haja um planejamento de políticas públicas para LV no Estado de São Paulo, Brasil, novos casos têm sido notificados e se disseminado. O artigo objetiva discutir como o Centro de Controle de Zoonoses realiza suas atividades espacialmente em uma cidade endêmica, Presidente Prudente, no Estado de São Paulo. Os dados são da Secretaria Municipal de Saúde de Presidente Prudente, Instituto Adolfo Lutz e Instituto Brasileiro de Geografia e Estatística. Estimamos espacialmente a população canina por setor censitário e utilizamos ferramentas de geoprocessamento para produzir mapas coropléticos, tendências espaciais e autocorrelação espacial. Encontramos um padrão espacial de maior prevalência na periferia da cidade e uma autocorrelação espacial positiva estatisticamente significativa (I = 0,2; p < 0,000) com clusters de relação alta-alta no noroeste da cidade. Além disso, identificamos uma direção diferente no caminho dos inquéritos sorológicos realizados versus a tendência na LV canina, o que enfatiza a fragilidade das medidas de controle do Centro de Controle de Zoonoses para controlar casos da doença. O Centro de Controle de Zoonoses parece estar sempre correndo atrás da doença. A análise espacial pode ser útil para repensar o funcionamento do serviço e auxiliar as políticas públicas.

La leishmaniasis visceral (LV) es un problema de salud pública en las ciudades brasileñas. Aunque hay políticas públicas de planificación relacionadas con la LV en el estado de São Paulo, Brasil, se han informado de nuevos casos, además de su propagación. El objetivo de este trabajo es discutir cómo el Centro de Control de Zoonosis dirige sus acciones espacialmente en una ciudad endémica del estado de São Paulo, Presidente Prudente. Los datos proceden de la Secretaría Municipal de Salud de Presidente Prudente, del Instituto Adolfo Lutz, y del Instituto Brasileño de Geografía y Estadística. Estimamos espacialmente la población de perros por sector censal y utilizamos herramientas de geoprocesamiento para elaborar mapas de coropletas, tendencias espaciales, y autocorrelación espacial. Encontramos un patrón espacial de más alta prevalencia en la periferia de la ciudad, además de una autocorrelación espacial positiva y estadísticamente significativa (I = 0,2; valor de p < 0,000) con clústeres de relaciones alto-alto en la parte noroccidental de la ciudad. Además, identificamos una dirección diferente en la trayectoria de las encuestas serológicas llevadas a cabo, frente a la tendencia de LV canina, que enfatiza la debilidad de acciones del Centro de Control de Zoonosis para controlar casos de la enfermedad. El Centro de Control de Zoonosis parece siempre estar tras la enfermedad. El análisis espacial podría ser útil para repensar cómo está funcionando el servicio, además de ayudar a políticas públicas.

Visceral leishmaniasis in an environmentally protected area in southeastern Brazil: Epidemiological and laboratory cross-sectional investigation of phlebotomine fauna, wild hosts and canine cases.

BACKGROUND: Leishmaniasis is a rapidly expanding zoonosis that shows increasing urbanization. Concern exists regarding the role of wildlife in visceral leishmaniasis (VL) transmission, due to frequent natural or anthropogenic environmental changes that facilitate contact between wildlife, humans and their pets. The municipality of Campinas, in southeastern Brazil, initially recorded VL in 2009, when the first autochthonous case was confirmed in a dog living in an upscale residential condominium, located inside an environmentally protected area (EPA). Since then, disease transmission remains restricted to dogs inhabiting two geographically contiguous condominiums within the EPA. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional study of the VL focus to investigate Leishmania spp. infection in domestic dogs, wild mammals and sand flies using molecular tools and recommended serological techniques. Canine seroprevalences of 1.5% and 1.2% were observed in 2013 and 2015, respectively. Six insect species, confirmed or suspected vectors or potential transmitters of Leishmania, were identified. Two specimens of the main L. (L.) infantum vector in Brazil, Lutzomyia longipalpis, were captured in the EPA. Natural infection by L. (L.) infantum was recorded in one Expapillata firmatoi specimen and two Pintomyia monticola. Natural infection by L. (L.) infantum and Leishmania subgenus Viannia was also detected in two white-eared opossums (Didelphis albiventris), a known reservoir of VL. Geographical coordinates of each sampling of infected animals were plotted on a map of the EPA, demonstrating proximity between these animals, human residences, including the dogs positive for VL, and forest areas. CONCLUSIONS/SIGNIFICANCE: The EPA, which is inhabited by humans, has an active VL focus. The risk of establishing and maintaining disease transmission foci in similar scenarios, i.e. wild areas that undergo environmental modifications, is evident. Moreover, different epidemiological profiles of VL must be included to elaborate prevention and control measures that consider the particularities of each transmission area.

Value of the oral swab for the molecular diagnosis of dogs in different stages of infection with Leishmania infantum.

This study was based on the need to employ a sensitive and specific method with samples that could be easily collected for diagnosing dogs infected with Leishmania infantum. To this end, we used real time-PCR (qPCR) to assess the value of the oral swab (OS) in detecting infected sick dogs (SD; n=62), including, for the first time, the analysis of apparently healthy infected dogs (AD; n=30), both from endemic areas for visceral leishmaniasis (VL). For comparison, we also evaluated the performance of the conjunctival swab (CS), blood (BL), lymph node (LN) and serology. We detected the presence of Leishmania DNA in the oral cavity in 62 out of the 92 dogs studied. The OS positivity (67.4%) was equivalent to the CS (68.5%) (p>0.05), higher than BL (52.2%) (p≤0.05), and lower than LN (84.8%) (p≤0.05). OS and CS performed well in SD dogs (82.3% and 83.9%, respectively) but not in AD dogs (36.7% for both samples). BL showed the lowest positivity (52.2%) and provided equivalent results between AD (60.0%) and SD (48.4%) dogs (p>0.05). LN yielded the highest positivity (84.8%), and it was also higher in the SD population (93.5%) compared to the AD population (66.7%) (p≤0.05). Parasite load was high in LN, moderate in OS and CS, and low in BL, showing the relationship between the levels of parasitism and the positivity rates found in these samples. Serology was positive in 82.2% of the SD group and in 70% of the AD dogs (p>0.05). Among the 20 seronegative dogs, seven (35%) were positive in either OS or CS, and 12 (60%) were positive when both noninvasive samples were jointly considered. The OS/CS combination resulted in a significant increase of positivity (p≤0.05) for the AD dogs (from 36.7% to 63.4%), as well as OS/serology (80%) and OS/CS/serology (83.4%). For the SD population, positivity reached up to 95.2% with the same combinations, showing that combination of samples and/or tests is required for the identification of dogs infected with L. infantum and that the OS and CS combination based on qPCR notably improves the detection of both AD and SD dogs. In conclusion, OS proved to be a suitable sample for the molecular diagnosis of infected dogs with clinical signs of VL, but not for dogs with inapparent infection. For these, we recommend the combination of OS results with CS and/or serology in order to reach relevant positivity for L. infantum. Finally, another advantage of using OS or both noninvasive samples is the increased likelihood of diagnosing seronegative dogs.

Serological, parasitological and molecular tests for canine visceral leishmaniosis diagnosis in a longitudinal study.

One of the measures to control visceral leishmaniosis (VL) in Brazil is the identification and culling of the canine reservoir. There is much controversy concerning this strategy, including the proper identification of positive dogs and the fact that the host-parasite relationship changes over time make it more challenging. A dynamic cohort of 62 dogs was followed every three months using serological and parasitological examinations and PCR. Positivity by PCR was higher than by serology and by parasitological examinations and showed a tendency to decrease over time, while serology tended to increase after six months. Concomitant positivity in all tests was observed in 10.4% of the samples, and negativity in 29.1%. Overall sensitivity ranged from 43.6 to 64.1%, and was not uniform over time. The proportion of dogs with or without clinical signs was not different by cytology or PCR but PCR was able to identify a larger number of asymptomatic dogs compared to ELISA and immunochromatography. PCR can be useful for surveillance of areas where cases of canine VL have not yet been detected and in which control strategies can be implemented to limit the spread of the disease. Despite the advance in diagnostic tools CVL diagnosis remains a challenge.