In silico and in vitro evaluation of primers for molecular differentiation of Leishmania species.

Leishmaniasis is a zoonotic disease caused by over 20 species of protozoan parasites of the genus Leishmania. Infection is commonly spread by sandflies and produces a wide spectrum of clinical signs and symptoms. Therefore, from an epidemiological and therapeutic standpoint, it is important to detect and differentiate Leishmania spp. The objective of this study was to combinate in silico and in vitro strategies to evaluate the analytical specificity of primers previously described in the literature. According to electronic PCR (e-PCR) analysis, 23 out of 141 pairs of primers selected through literature search matched their previously reported analytical specificity. In vitro evaluation of nine of these primer pairs by quantitative PCR (qPCR) confirmed the analytical specificity of five of them at the level of Leishmania spp., L. mexicana complex or Leishmania and Viannia subgenera. Based on these findings, the combination of e-PCR and qPCR is suggested to be a valuable approach to maximize the specificity of new primer pairs for the laboratory diagnosis of infections with Leishmania spp.

In silico and in vitro evaluation of primers for molecular differentiation of Leishmania species / Avaliação in silico e in vitro de iniciadores para a diferenciação molecular de espécies de Leishmania

Abstract Leishmaniasis is a zoonotic disease caused by over 20 species of protozoan parasites of the genus Leishmania. Infection is commonly spread by sandflies and produces a wide spectrum of clinical signs and symptoms. Therefore, from an epidemiological and therapeutic standpoint, it is important to detect and differentiate Leishmania spp. The objective of this study was to combinate in silico and in vitro strategies to evaluate the analytical specificity of primers previously described in the literature. According to electronic PCR (e-PCR) analysis, 23 out of 141 pairs of primers selected through literature search matched their previously reported analytical specificity. In vitro evaluation of nine of these primer pairs by quantitative PCR (qPCR) confirmed the analytical specificity of five of them at the level of Leishmania spp., L. mexicana complex or Leishmania and Viannia subgenera. Based on these findings, the combination of e-PCR and qPCR is suggested to be a valuable approach to maximize the specificity of new primer pairs for the laboratory diagnosis of infections with Leishmania spp.

Resumo As leishmanioses são zoonoses causadas por mais de 20 espécies de protozoários do gênero Leishmania. As infecções são comumente disseminadas por flebotomíneos e causam um amplo espectro de manifestações clínicas. Portanto, a detecção e diferenciação de espécies de Leishmania são importantes do ponto de vista epidemiológico e terapêutico. O objetivo deste estudo foi combinar estratégias in silico e in vitro para avaliar a especificidade analítica dos primers descritos anteriormente na literatura. De acordo com a PCR eletrônica (e-PCR), 23 dos 141 pares de primers selecionados por meio de pesquisa da literatura estavam de acordo com a especificidade analítica anteriormente relatada. A avaliação in vitro de nove desses pares de primers, por PCR quantitativa (qPCR), confirmou a especificidade analítica de cinco deles ao nível de espécie de Leishmania, do complexo L. mexicana ou dos subgêneros Leishmania e Viannia. Com base nos resultados, sugere-se que a combinação de e-PCR e qPCR é uma abordagem valiosa para a validação e maximização da especificidade de novos pares de primers para o diagnóstico laboratorial de infecções com Leishmania spp.

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.

[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.

Controle da leishmaniose visceral canina por eutanásia: estimativa de efeito baseado em inquérito e modelagem matemática / Control of canine visceral leishmaniasis by euthanasia: estimated effect based on a survey and mathematical modeling / Control de la leishmaniasis visceral canina por eutanasia: estimación del efecto basado en una encuesta y modelo matemático

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.

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.

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.

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.

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.

Factors associated with positivity for canine visceral leishmaniosis in an endemic area in Brazil.

Visceral leishmaniosis (VL) is a public health problem and its occurrence depends primarily on the presence of the vector and susceptible hosts; in the urban environment, the dog is the main reservoir. This study aimed to analyze the distribution of canine visceral leishmaniosis (CVL) and factors associated with it in an urban area endemic for VL. Analysis of the variables was based on 2755 epidemiological records of dogs positive and negative for CVL over a three-year period (2009, 2010 and 2011). A dog was considered positive when it presented amastigotes in the cytological examination of lymph node biopsy and/or was seropositive by immunoenzymatic and indirect immunofluorescence assays. CVL positive dogs were observed throughout the town, but significant differences were observed between the sectors analyzed (P<0.0001), with two sectors showing higher positivity. CVL prevalence was 35.9% and was significantly associated with age and breed (P<0.0001). Concerning symptoms, 44.3% of symptomatic dogs were positive for LV (P<0.0001) in an urban area endemic for this zoonosis.

Human visceral leishmaniasis and relationship with vector and canine control measures / Leishmaniose visceral em humanos e relação com medidas de controle vetorial e canino

ABSTRACT 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.

RESUMO OBJETIVO Estimar a cobertura das atividades de controle da leishmaniose visceral e relacioná-las com a ocorrência de leishmaniose visceral em humanos em área urbana endêmica. MÉTODOS Foram considerados como população de estudo os casos de leishmaniose visceral em humanos e em cães avaliados por inquérito sorológico censitário realizado em Araçatuba, SP, de 2007 a 2015. Os casos de leishmaniose visceral em humanos foram geocodificados pelo endereço de residência dos pacientes e, os cães, pelo endereço de residências dos respectivos tutores. Foram calculadas as coberturas do inquérito sorológico, da eutanásia e de borrifação de inseticida, as soroprevalências caninas e as taxas de incidência de leishmaniose visceral em humanos. A relação entre a leishmaniose visceral em humanos e as medidas de controle, bem como a soroprevalência foram avaliadas por comparação de mapas e por meio de regressão linear. A relação entre a doença canina e a humana também foi avaliada por meio da função K de Ripley . RESULTADOS As taxas de incidência de leishmaniose visceral em humanos apresentaram um período de declínio (2007 a 2009) e um período de estabilidade (2010 a 2015), comportamento semelhante ao das soroprevalências caninas. Em geral, a cobertura das medidas de controle foi baixa e a não associação com a incidência de leishmaniose visceral em humanos pode ser consequência do período analisado e do número pequeno de unidades analisadas (setores da Superintendência de Controle de Endemias). A distribuição dos casos humanos apresentou dependência espacial com a distribuição dos cães soropositivos de 2007 a 2009. CONCLUSÕES Este trabalho reafirmou a relação entre a ocorrência da doença no homem e no cão, verificou a diminuição das taxas de leishmaniose visceral em humanos e em cães em Araçatuba ao longo do tempo, mesmo em baixa cobertura das atividades de controle. Entretanto, novos estudos são necessários para averiguar se fatores além das atividades de vigilância e controle estariam envolvidos na diminuição das incidências.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines.

Plasma HPV DNA is detectable in oral leukoplakia patients.

BACKGROUND: Oral leukoplakia is considered a potentially malignant lesion for the development of squamous cell carcinoma, and various risk factors may be related to its development and malignant transformation, including the human papillomavirus (HPV). The aim of this case-control study was to detect the presence of HPV in fresh tissue, plasma and saliva samples obtained from patients with and without oral leukoplakia, and verify the correlation of the presence of DNA of HPV between different sources of materials. METHODS: In this study, 32 patients with oral leukoplakia and 24 patients selected in a case-control manner were included. DNA extraction from the samples was performed, and afterwards it was amplified by nested polymerase chain reaction (nPCR) for the detection of HPV (nPCR: MY09-MY11/GP05+-GP06+). RESULTS: The DNA of HPV was found in 68.75% of the fresh tissue samples; in 50% of plasma, and in 62.5% of saliva samples in the group of patients with leukoplakia; in comparison with 45.8%, 54%, and 45.8%, in the fresh tissue, plasma and saliva samples, respectively, in the control group. CONCLUSION: Based on the present study, there was no difference in the rate of HPV detection in patients with or without oral leukoplakia. However all sources tested in this study were considered suitable for HPV detection, especially plasma samples, which showed be an important non-invasive source of HPV detection in leukoplakia patients.

Prediction of lymph node parasite load from clinical data in dogs with leishmaniasis: An application of radial basis artificial neural networks.

Quantification of Leishmania infantum load via real-time quantitative polymerase chain reaction (qPCR) in lymph node aspirates is an accurate tool for diagnostics, surveillance and therapeutics follow-up in dogs with leishmaniasis. However, qPCR requires infrastructure and technical training that is not always available commercially or in public services. Here, we used a machine learning technique, namely Radial Basis Artificial Neural Network, to assess whether parasite load could be learned from clinical data (serological test, biochemical markers and physical signs). By comparing 18 different combinations of input clinical data, we found that parasite load can be accurately predicted using a relatively small reference set of 35 naturally infected dogs and 20 controls. In the best case scenario (use of all clinical data), predictions presented no bias or inflation and an accuracy (i.e., correlation between true and predicted values) of 0.869, corresponding to an average error of ±38.2 parasites per unit of volume. We conclude that reasonable estimates of L. infantum load from lymph node aspirates can be obtained from clinical records when qPCR services are not available.

Age structure of owned dogs under compulsory culling in a visceral leishmaniasis endemic area.

The age structure of the dog population is essential for planning and evaluating control programs for zoonotic diseases. We analyzed data of an owned-dog census in order to characterize, for the first time, the structure of a dog population under compulsory culling in a visceral leishmaniasis endemic area (Panorama, São Paulo State, Brazil) that recorded a dog-culling rate of 28% in the year of the study. Data on 1,329 households and 1,671 owned dogs revealed an owned dog:human ratio of 1:7. The mean age of dogs was estimated at 1.73 years; the age pyramid indicated high birth and mortality rates at the first year of age with an estimated cumulative mortality of 78% at the third year of age and expected life span of 2.75 years. In spite of the high mortality, a growth projection simulation suggested that the population has potential to grow in a logarithmic scale over the years. The estimated parameters can be further applied in models to maximize the impact and minimize financial inputs of visceral leishmaniasis control measures.

Spinal Taenia solium cysticercosis in Mexican and Indian patients: a comparison of 30-year experience in two neurological referral centers and review of literature.

OBJECTIVE: To present a retrospective study from patients with spinal cysticercosis (SC), diagnosed within the last 30 years in Mexican and Indian neurological referral centers. METHODS: This is a retrospective and comparative study of the clinical and radiological profile between Mexican and Indian patients with spinal neurocysticercosis during a 30-year period and a review of the literature during the same period. RESULTS: Twenty-seven SC patients were included: 19 from Mexico and 8 from India. SC presented predominantly with motor symptoms (21/27 patients): paraparesis and paraplegia were the most common signs; one-third of patients presented sphincter dysfunction. Imaging studies showed that parasites in vesicular stage were more frequent in patients from Mexico, while degenerative stages predominated in India. Association of subarachnoid cysticerci and hydrocephalus was observed only in Mexican patients. CONCLUSIONS: Despite the limitations of this study, the collected information supports the existence of differences in the clinical and radiological traits of SC patients between Asian and Latin-American hospitals. The possible biological factors that may underlie these differences are discussed.

Detection of Epstein-Barr virus in different sources of materials from patients with oral lichen planus: a case-control study.

AIMS: To detect the presence of Epstein-Barr virus (EBV) DNA in different sources of materials from a matched group of patients with oral lichen planus (OLP) and a group of people without OLP lesions, and to correlate the presence of virus with epidemiological variables of the groups studied. METHODS: Fresh tissue samples, saliva, exfoliated cells and plasma of 24 patients with OLP lesions (cases) and 17 patients without OLP lesions (controls) were collected. EBV was detected by nested PCR. RESULTS: Viral positivity was obtained in 62.5% of tissue samples; in 70.8% of exfoliated cell samples; in 33.3% of blood plasma samples and in 75% of saliva samples in the cases; and in 35.3% of tissue samples; 82.4% of exfoliated cell samples; in 47.1% of blood plasma samples and in 64.7% of saliva samples in the controls. There was a predominance of women in both groups. Variables not atrophic-erosive were most affected by EBV. CONCLUSIONS: No relationship between EBV and OLP was found. However, all sources tested in this study were considered suitable for the detection of viruses.

Age structure of owned dogs under compulsory culling in a visceral leishmaniasis endemic area / A estrutura etária de cães com donos numa área endêmica de leishmaniose visceral / Estructura etaria de la población canina domiciliada en un área endémica con leishmaniasis visceral canina

Abstract: The age structure of the dog population is essential for planning and evaluating control programs for zoonotic diseases. We analyzed data of an owned-dog census in order to characterize, for the first time, the structure of a dog population under compulsory culling in a visceral leishmaniasis endemic area (Panorama, São Paulo State, Brazil) that recorded a dog-culling rate of 28% in the year of the study. Data on 1,329 households and 1,671 owned dogs revealed an owned dog:human ratio of 1:7. The mean age of dogs was estimated at 1.73 years; the age pyramid indicated high birth and mortality rates at the first year of age with an estimated cumulative mortality of 78% at the third year of age and expected life span of 2.75 years. In spite of the high mortality, a growth projection simulation suggested that the population has potential to grow in a logarithmic scale over the years. The estimated parameters can be further applied in models to maximize the impact and minimize financial inputs of visceral leishmaniasis control measures.

Resumo: É importante conhecer a estrutura etária da população canina para melhor planejar e avaliar os programas de controle de zoonoses. Os autores analisaram os dados de um censo de cães com donos para caracterizar, pela primeira vez, a estrutura etária de uma população canina sujeita à eliminação compulsória numa área endêmica de leishmaniose visceral (Panorama, Estado de São Paulo, Brasil) que registrou uma taxa de eliminação canina de 28% no ano do estudo. Os dados para 1.329 domicílios e 1.671 cães com donos mostraram uma razão de cães para humanos de 1:7. A média de idade dos cães foi estimada em 1,73 anos; a pirâmide etária indicou altas taxas de natalidade e de mortalidade no primeiro ano de vida, com uma estimativa de mortalidade cumulativa de 78% aos três anos de idade, e uma expectativa de vida de 2,75 anos. Apesar da alta mortalidade, uma simulação de projeção de crescimento sugeriu que a população tem o potencial de crescer numa escala logarítmica ao longo dos anos. Os parâmetros estimados podem ser aplicados também a modelos para maximizar o impacto e minimizar os insumos financeiros de medidas de controle da leishmaniose visceral.

Resumen: El conocimiento de la estructura etaria de una población de perros es esencial para la planificación de programas de control de zoonosis. Se analizaron datos de un censo de población canina domiciliada, con el objetivo de caracterizar, por primera vez, la estructura de una población de perros domiciliados en un área donde la eutanasia de perros positivos en leishmaniasis visceral es obligatoria (Panorama, São Paulo, Brasil), y que registró un 28% de casos de eutanasia en el año en que el censo fue realizado. Los datos de 1.329 domicilios y 1.671 perros resultaron en una razón perro:hombre de 1:7. La edad media de los perros fue estimada en 1,73 años; la pirámide de edad indica altas tasas de nacimiento y mortalidad hasta 1 año de vida, con tasa de mortalidad acumulada de un 78% en el tercer año de vida, y expectativa de vida de 2,75 años. A pesar de la alta tasa de mortalidad, la simulación de crecimiento poblacional sugiere que esta población tiene potencial de crecimiento en escala logarítmica a lo largo de los años. Los parámetros estimados pueden ser utilizados en modelos para maximizar el impacto y minimizar los costes de las medidas de control de la enfermedad.

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.

Serological, parasitological and molecular tests for canine visceral leishmaniosis diagnosis in a longitudinal study / Testes sorológicos, parasitológicos e moleculares para o diagnóstico da leishmaniose visceral canina em estudo longitudinal

Abstract 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.

Resumo Uma das medidas de controle da leishmaniose visceral (LV) no Brasil se baseia na identificação e eliminação do reservatório canino. Existe considerável controvérsia relativa a esta estratégia incluindo a correta identificação dos cães positivos e a variação temporal da relação hospedeiro-parasita, o que torna esta medida ainda mais desafiadora. Uma coorte dinâmica de 62 cães foi acompanhada trimestralmente utilizando-se métodos sorológicos, parasitológicos e a PCR. A taxa de positividade por PCR foi maior em comparação à dos métodos sorológicos e parasitológicos, e mostrou tendência à diminuição com o passar do tempo, enquanto que a positividade sorológica apresentou tendência a aumento, após seis meses. Observou-se positividade concomitante em todos os testes em 10,4% das amostras e, negatividade concomitante, em 29,1%. A sensibilidade geral variou de 43,6% a 64,1%, não sendo uniforme ao longo do estudo. A proporção de cães com e sem sinais clínicos que foram positivos ao exame parasitológico ou à PCR não foi estatisticamente diferente. Contudo, foi possível identificar como positivos um maior número de animais assintomáticos por meio da técnica de PCR, em comparação aos testes ELISA e imunocromatográfico. A PCR pode ser bastante útil para a vigilância epidemiológica de áreas onde casos de LV canina ainda não tenham sido descritos e onde estratégias de controle podem ser implantadas para limitar a disseminação da doença. Não obstante o avanço nas ferramentas diagnósticas, diagnosticar a LVC continua um desafio.

First detection of Leishmania spp. DNA in Brazilian bats captured strictly in urban areas.

Leishmania spp. is a protozoan that maintains its life cycle in domestic and wild animals and it may include bats, a population that has increased in urban environments. This study aimed to investigate the presence of Leishmania spp. in bats captured strictly in urban areas that are endemic for visceral leishmaniasis. The spleen and skin samples of 488 bats from 21 endemic cities in northwestern São Paulo State, Brazil, were tested for the presence of Leishmania kDNA using real-time PCR. Differentiation from Trypanosoma spp. was achieved by amplifying a DNA fragment of the ribosomal RNA gene. The presence of Leishmania spp. kDNA was verified in 23.9% of bats and Trypanosoma spp. DNA was identified in 3.9%. Leishmania species differentiation revealed the presence of Leishmania amazonensis in 78.3% of the bats; L. infantum in 17.4%, and 1 sample (4.3%) showed a mix pattern of L. infantum and L. amazonensis. We also detected, for the first time, L. infantum and L. amazonensis DNA in Desmodus rotundus, the hematophagous bat. The presence of Leishmania spp. DNA in bats strictly from urban areas endemic for visceral leishmaniasis in the State of São Paulo, Brazil indicates that these wild and abundant animals are capable of harboring Leishmania spp. in this new scenario. Due to their longevity, high dispersion capacity and adaptability to synanthropic environments, they may play a role in the maintenance of the life cycle of Leishmania parasites.

Usefulness of serological ELISA assay for Taenia saginata to detect naturally infected bovines.

Bovine cysticercosis, a cosmopolitan disease caused by Taenia saginata, leads to economic losses due to carcass devaluation at slaughter. Sanitary inspection at slaughterhouses, the routine diagnostic method in Brazil, lacks the necessary sensitivity to detect the mildly infected cattle that are typically encoutered in Brazil. In this study we have tested cattle sera from animals diagnosed as positive and negative by veterianry inspection for (1) anti-parasite antibodies using metacestodes antigens (T. solium vesicular fluid and T. saginata secretions) and (2) the HP10 secreted antigen of viable metacestodes. The cut-off values were calculated by ROC curve for intense and mild infections conditions, and by the classical method ( for negative samples). The sensitivity and specificity of these diagnostic tests were different depending on the assumed cut-off value and, importantly, whether the infection was mild or intense. In spite of these observations, however, such ELISA assays for serum antibodies and parasite antigens constitute an important tool for epidemiological porposes, and in establishing priorities for the control of bovine cysticercosis.