Scoring clinical signs can help diagnose canine visceral leishmaniasis in a highly endemic area in Brazil

Canine visceral leishmaniasis (CVL) diagnosis is still a challenge in endemic areas with limited diagnostic resources. This study proposes a score with the potential to distinguish positive CVL cases from negative ones. We studied 265 dogs that tested positive for CVL on ELISA and parasitological tests. A score ranging between 0 and 19 was recorded on the basis of clinical signs. Dogs with CVL had an overall higher positivity of the majority of clinical signs than did dogs without CVL or with ehrlichiosis. Clinical signs such as enlarged lymph nodes (83.93%), muzzle/ear lesions (55.36%), nutritional status (51.79%), bristle condition (57.14%), pale mucosal colour (48.21%), onychogryphosis (58.93%), skin lesion (39.28%), bleeding (12.50%), muzzle depigmentation (41.07%), alopecia (39.29%), blepharitis (21.43%), and keratoconjunctivitis (42.86%) were more frequent in dogs with CVL than in dogs with ehrlichiosis or without CVL. Moreover, the clinical score increased according to the positivity of all diagnostic tests (ELISA, p < 0.001; parasite culture, p = 0.0021; and smear, p = 0.0003). Onychogryphosis (long nails) [odds ratio (OR): 3.529; 95% confidence interval (CI): 1.832-6.796; p < 0.001], muzzle depigmentation (OR: 4.651; 95% CI: 2.218-9.750; p < 0.001), and keratoconjunctivitis (OR: 5.400; 95% CI: 2.549-11.441; p < 0.001) were highly associated with CVL. Interestingly, a score cut-off value ≥ 6 had an area under the curve of 0.717 (p < 0.0001), sensitivity of 60.71%, and specificity of 73.64% for CVL diagnosis. The clinical sign-based score for CVL diagnosis suggested herein can help veterinarians reliably identify dogs with CVL in endemic areas with limited diagnostic resources.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test / Expressão de uma proteína recombinante, da família A2, de Leishmania infantum (amostra Jaboticabal) e sua avaliação no teste sorológico da Leishmaniose Visceral Canina

This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

Este estudo teve como objetivos expressar uma proteína recombinante da família A2 de Leishmania chagasi, amostra de Jaboticabal-SP; testar essa proteína como antígeno em testes sorológicos; e investigar a antigenicidade e imunogenicidade dessa proteína. Uma proteína codificada por um alelo do gene A2 isolado de L. chagasi foi expressa em três diferentes amostras de Escherichia coli. Foram utilizadas 29 amostras de soro de cães vacinados com Leishmune, 482 amostras de soro de cães de áreas endêmicas (controles positivos), e 170 amostras de soro de cães de áreas não-endêmicas (controles negativos) no ELISA-teste utilizando-se antígeno solúvel total de Leishmania (AST) e His-A2 como antígenos. As proteínas expressas, detectadas pelo western blotting, mostraram a expressão de uma proteína de 11 KDa. Sessenta e três por cento (303/482) das amostras de áreas endêmicas foram positivas pelo ELISA-teste, utilizando-se antígeno His-A2; e 93,1% (27/29) dos animais vacinados com a Leishmune foram negativos. Anticorpos anti-A2 de camundongos inoculados com a proteína A2 foram detectados em lâminas contendo formas amastigotas, enquanto em lâminas contendo formas promastigotas não houve detecção de anticorpos anti-A2. A proteína recombinante A2 pode ser uma ferramenta útil no diagnóstico da LVC, e maiores estudos sobre o estágio de infecção e a espécie de parasita dos cães amostrados devem prover melhor entendimento dos resultados encontrados.

Field validation of a Leishmania (Leishmania) mexicana exo-antigens ELISA for diagnosing tegumentary leishmaniasis in regions of Leishmania (Viannia) predominance

Background: Several tests are performed to obtain better accuracy when diagnosing American tegumentary leishmaniasis (ATL). It is believed that antigens released via secretion, excretion and metabolism are more specific than are antigens released by the lysis of Leishmania parasites. Such antigens are known as exo-antigens (exo-Ag) and are formed from products released by cultured parasites in a way that is similar to that in which they cause infections in hosts. Objective: We attempted to validate a Leishmania mexicana ELISA exo-Ag for ATL diagnosis in Midwestern Brazil. Methods: A total of 281 patients were included in the study. We analysed pre-treatment blood from 98 ATL patients; out of those, 85.7% and 14.3% had cutaneous and mucosal forms, respectively. Results: The exo-Ag accuracy was 83.99% (95% CI = 79.24-87.81) with a sensitivity value of 90.82% (95% CI = 83.46-95.09) and an overall specificity value of 80.33% (95% CI = 73.97-85.44). The positive predictive value and negative predictive value were 71.20% (95% CI = 62.72-78.41) and 94.23% (95% CI = 89.40-96.94), respectively. Among healthy controls, exo-Ag had a specificity of 91.25% (95% CI = 83.02-95.70); additionally, the test had specificity rates of 66.67% (95% CI = 46.71-82.03) in Chagas disease patients, 60.61% (95% CI = 43.68-75.32) in patients with rheumatic diseases, 76.92% (95% CI = 49.74-91.82) in pemphigus foliaceus patients, 87.50% (95% CI = 52.91-97.76) in leprosy patients, 87.50% (95% CI = 63.98-96.50) in VRDL-positive patients, and 77.78 (95% CI = 45.26-93.68) in deep mycosis patients. Conclusion: Based on the indicators of validity, we conclude that the results obtained in this study enable the recommendation of the exo-Ag ELISA for ATL diagnosis once it presented a reasonable accuracy compared to classical methods. Cost evaluations are necessary to completely define the role of this technique in large scale. .

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs / Utilização de um ELISA baseado em NcSRS2 de Neospora caninum expressa em Pichia pastoris para diagnóstico de neosporose em ovinos e cães

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.

PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL / Prevalência de Entamoeba histolytica/Entamoeba dispar na cidade de Campina Grande, Nordeste do Brasil

There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species.

A prevalência mundial de Entamoeba histolytica não está bem estabelecida. Este fato deve-se à complicação derivada da existência de duas espécies morfologicamente idênticas, mas geneticamente diferentes: a E. histolytica que causa amebíases e a E. dispar descrita como não patogênica. No Brasil, em comunidades com precárias condições sanitárias e endêmicas para várias parasitoses, localizadas nas regiões Sudeste (SE) e Nordeste (NE), somente E. dispar tem sido encontrada, porém outras regiões, apresentam indivíduos infectados por E. histolytica. Na região agreste do Estado da Paraíba (NE) que apresenta as mesmas precárias condições sanitárias, não tem sido reportada prevalência específica destes parasitos, embora fosse encontrada alta prevalência do complexo E. dispar/E. histolytica em crianças em favela urbana. O presente estudo foi realizado em favela da cidade de Campina Grande, Estado da Paraíba, onde 1.195 crianças de dois a 10 anos sem sintomatologia foram examinadas. Amostras de fezes destas crianças foram analisadas microscopicamente, encontrando-se 553 positivas para o complexo E. dispar/E. histolytica. Do total de amostras positivas, 456 foram submetidas à pesquisa do antígeno especifico para E. histolytica pelo teste ELISA E. histolytica II®,obtendose resultado negativo para a presença do antígeno adesina específico de E. histolytica, em todas as amostras testadas. Os resultados sugerem que nesta comunidade não há infecção por E. histolytica, e que E. dispar é a espécie dominante na região.

Soroepidemiologia de Toxoplasma gondii em idosos atendidos pela Estratégia Saúde da Família, Porto Alegre, Rio Grande do Sul, Brasil / Seroepidemiology of Toxoplasma gondii in elderly individuals treated under the Family Health Strategy, Porto Alegre, Rio Grande do Sul, Brazil

O objetivo desse estudo foi avaliar a soroprevalência para Toxoplasma gondii e a relacionar com as condições socioeconômicas, higiênicas, sanitárias e de saúde nos idosos da Estratégia Saúde da Família - ESF, do município de Porto Alegre, Rio Grande do Sul, Brasil. A pesquisa realizada foi um estudo transversal, no qual foi aplicado um questionário de inquérito epidemiológico e realizada coleta de sangue. A avaliação de IgG e IgM anti-T. gondii foi realizada pela técnica de ELISA. Foram avaliados 599 idosos com soroprevalência para IgG anti-T. gondii de 88,0% e de 0,8% para IgM. Na análise multivariada, as variáveis que se associaram de forma independente para IgG positivo foram: faixa etária, renda pessoal e uso de óculos; e para IgM positivo: faixa etária, autopercepção de saúde e uso de óculos. Os resultados obtidos chamam a atenção pela alta prevalência de IgG anti-T. gondii nos idosos da ESF de Porto Alegre, gerando uma preocupação no caso de ocorrência de reativação da toxoplasmose e desenvolvimento dos sintomas mais graves dessa infecção.

The aim of this study was to evaluate the seroprevalence of Toxoplasma gondii and relate it to the socioeconomic, hygienic, sanitary and health conditions of the elderly of the Family Health Strategy (FHS) in the city of Porto Alegre, Rio Grande do Sul, Brazil. The research involved a cross-sectional study in which a questionnaire with epidemiologic questions was applied and blood samples were taken. The assessment of IgG and IgM anti-T. gondii was performed using the ELISA technique. Seroprevalence was evaluated among 599 elderly individuals with 88% for IgG anti-T. gondii and with 0.8% for IgM. In the multivariate analysis, the variables that associated themselves independently with positive IgG were age range, personal income and wearing spectacles. Those associated with positive IgM were age, self-rated health and wearing spectacles. The results call attention to the high prevalence of IgG anti-T. gondii in elderly individuals in the FHS in Porto Alegre, generating concern in the event that the reactivation of toxoplasmosis and the development of more severe symptoms of this infection occur.

Seroprevalence and major antigens recognized by sera from Trypanosoma cruzi-infected dogs from Jalisco, México / Seroprevalencia y principales antígenos reconocidos por sueros de perros infectados con Trypanosoma cruzi en el estado de Jalisco, México

Chagas disease is a major endemic disease caused by the protozoan parasite Trypanosoma cruzi. This parasitic disease is widely distributed throughout Latin America, affecting 10 million people. There are also reports of canine infection in the southern part of the United States. Dogs are considered the predominant domestic reservoir for T. cruzi in many areas of endemicity. In México, dog infection by this parasite has been poorly studied. In this work 209 dogs from six villages in Jalisco, México, were assessed to detect anti-T. cruzi antibodies by ELISA and Western blot. Seventeen (17) seropositive dogs (8.1 %) were detected by both tests, representing a seropositive value similar to that found in some southern states of México where the infection is present. No statistical differences were observed concerning the age and sex of infected and non-infected dogs. The major antigens recognized by positive sera were 26, 32, 66 and 80 kDa. These proteins are candidates to develop a specific diagnostic method for canine Chagas. No antibodies against HSP16 protein were found in T. cruzi seropositive sera. This is the first report of canine serology of Chagas disease in this central part of México. This report will contribute to the knowledge of the infection status of domestic reservoirs in the state of Jalisco, México.

El mal de Chagas es una enfermedad endémica causada por el parásito protozoario Trypanosoma cruzi. Este padecimiento está ampliamente distribuido en América, donde afecta a alrededor de 10 millones de personas. También existen comunicaciones de la infección canina desde el sur de los Estados Unidos hasta países de Sudamérica. Los perros son considerados los principales reservorios domésticos de T. cruzi en muchas áreas endémicas. En México, la infección canina ha sido estudiada escasamente. En el presente trabajo se evaluó mediante ELISA y Western blot la presencia de anticuerpos anti-T. cruzi en el suero de 209 perros de seis localidades del estado de Jalisco, México. Se encontraron 17 perros seropositivos (8,1 %) a ambas pruebas. No se observaron diferencias de significación estadística en la edad o el sexo de los perros infectados comparados con los no infectados. Los principales antígenos reconocidos por los sueros positivos fueron de 26, 32, 66 y 80 kDa. Estas proteínas son candidatos para desarrollar un método de diagnóstico específico para Chagas canino. No se encontraron anticuerpos contra la proteína HSP16 en los sueros positivos anti-T. cruzi. Este es el primer informe de serología canina en la región central de México y contribuirá al conocimiento de la infección en reservorios domésticos de Jalisco, México.

Comparison of Two Rapid Immunochromatographic Assays (ICT Malaria P.f. /P.v. Test and OptiMAL Test) with Microscopy for Detection of Malaria Parasites.

This study was done to compare the ability of newly developed immunochromatographic assays (ICT), i.e., ICT malaria P.f. / P.v. test and optiMAL test with standard microscopy for the diagnosis of malaria. ICT P.f. / P.v. test detects Plasmodium falciparum specific histidine rich protein-2 (HRP2) antigen and a pan-malarial common specific antigen, where as optiMAL test detects P. falciparum specific parasite Lactate Dehydrogenase (pLDH) enzyme and a common specific pLDH enzyme. Material and Methods: Blood samples were obtained from 150 patients clinically diagnosed as malaria between July 2011 to December 2011.The venous blood were tested for malaria by microscopy and simultaneously ICT P.f./P.v.and optiMAL tests. Results: From total 150 samples, 59 (39.3%) were positive by blood films while 64 (42.7%) were positive by ICT p.f. / p.v. and 52 (34.7%) by optiMAL tests. The blood film indicated that 32.2% (19 of 59) of patients were positive for P. vivax and 67.8% (40 of 59) were infected with P. falciparum. ICT P.f./P.v. test showed 23.4% (15 of 64) were positive for P. vivax and 76.6% (49 of 64) were infected with P. falciparum. Similarly, optiMAL test detected 30.8% (16 of 52) were positive for P. vivax and 69.2% (36 of 52) were infected with P. falciparum. ICT P.f./P.v. test had sensitivities 78.9%, 87.5% and specificities 100%, 87.3% for P. vivax and P. falciparum respectively. optiMAL test showed sensitivities 84.2%, 80% and specificities 100%, 96.4% for P. vivax and P. falciparum respectively. Conclusion: These rapid immunoassays (ICT P.f./P.v. and optiMAL) tests can be used as supplementary to traditional light microscopy for the diagnosis of malarial parasites.

Evaluation of the Accuracy of the EasyTest(TM) Malaria Pf/Pan Ag, a Rapid Diagnostic Test, in Uganda

In recent years, rapid diagnostic tests (RDTs) have been widely used for malaria detection, primarily because of their simple operation, fast results, and straightforward interpretation. The Asan EasyTest(TM) Malaria Pf/Pan Ag is one of the most commonly used malaria RDTs in several countries, including Korea and India. In this study, we tested the diagnostic performance of this RDT in Uganda to evaluate its usefulness for field diagnosis of malaria in this country. Microscopic and PCR analyses, and the Asan EasyTest(TM) Malaria Pf/Pan Ag rapid diagnostic test, were performed on blood samples from 185 individuals with suspected malaria in several villages in Uganda. Compared to the microscopic analysis, the sensitivity of the RDT to detect malaria infection was 95.8% and 83.3% for Plasmodium falciparum and non-P. falciparum, respectively. Although the diagnostic sensitivity of the RDT decreased when parasitemia was or =100 parasites/microl. The specificity of the RDT was 97.3% for P. falciparum and 97.3% for non-P. falciparum. These results collectively suggest that the accuracy of the Asan EasyTest(TM) Malaria Pf/Pan Ag makes it an effective point-of-care diagnostic tool for malaria in Uganda.

Evaluación de campo de la precisión de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia / Field evaluation for diagnostic accuracy of the rapid test SD Bioline Malaria Antigen Pf/Pv® in Colombia

Introducción. Las pruebas de diagnóstico rápido han sido postuladas como una forma de garantizar el diagnóstico de malaria, o paludismo, en zonas de difícil acceso. A pesar de su uso difundido, no hay estudios de campo que evalúen la precisión de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia. Objetivo. Evaluar la precisión diagnóstica de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv ®, en dos departamentos endémicos para malaria, comparando el diagnóstico con la gota gruesa corregida por reacción en cadena de la polimerasa (PCR). Materiales y métodos. Se trata de un estudio retrospectivo para evaluar sensibilidad, especificidad, valor diagnóstico positivo (VPP) y negativo (VPN), concordancia y límites de sensibilidad por rangos de parasitemia, de la prueba SD Bioline Malaria Antigen ® Pf/Pv, en Córdoba y Chocó. Los resultados fueron comparados con la gota gruesa corregida por PCR. Resultados. De 383 muestras procesadas, 121 fueron positivas (75 para Plasmodium vivax, 42 para P. falciparum y 4 para infección mixta) y 262 muestras negativas; los resultados obtenidos fueron los siguientes: P. vivax: sensibilidad, 92,0 % (IC 95% 83,6-96,3); especificidad, 98,7 % (IC 95% 96,7-99,5); VPP, 94,5 % (IC 95% 86,7-97,9); VPN, 98,1 % (IC 95% 95,8-99,1); IK, 0,90 (0,80-1,00). P. falciparum: sensibilidad, 88,1 % (IC 95% 75,0-94,8); especificidad, 97,9 % (IC 95% 95,8-99,0); VPP, 84,1% % (IC 95% 70,6-92,1); VPN, 98,5 % (IC 95% 96,6-99,4); IK, 0,80 (0,70-0,90). Conclusiones. La prueba tuvo un buen desempeño, siendo mejor para P. vivax en comparación con que para P. falciparum. Persisten dificultades en la detección de bajas parasitemias. La falta de amplificación de los genes Pfhrp2 y Pfhrp3 en dos muestras con diagnóstico de como infección mixta, sugiere una posible deleción conjunta de estos genes.

Introduction: Rapid diagnostic tests (RDT) have been postulated as a way to ensure access to malaria diagnosis in remote areas. Despite its widespread use, there are no field studies to evaluate the accuracy of the SD Bioline Malaria Antigen Pf/Pv in Colombia RDT. Objective: To evaluate the diagnostic accuracy of the SD Bioline Malaria Antigen Pf/Pv® RDT in two departments endemic for malaria, comparing diagnosis with thick film corrected with PCR. Materials and methods: A retrospective study was carried out to evaluate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), concordance and sensitivity limits according to parasitemia ranges for the SD Bioline Malaria Antigen Pf/Pv ® test in Cordoba and Choco. The results were compared with microscopy corrected by PCR. Results: A total of 383 samples processed, 121 were positive (75 for P. vivax , 42 for P. falciparum and 4 for mixed infection) and 262 negative samples. P. vivax: sensitivity 92.0% (95% CI: 83.6-96.3), specificity 98.7% ( 95% CI: 96.7-99.5), PPV 94.5% (95% CI: 86.7-97.9), NPV 98.1% (95% CI: 95.8-99.1), Cohen´s kappa coefficient was 0.90 (0.80-1.00). P. falciparum: sensitivity 88.1% (95% CI: 75.0-94.8), specificity 97.9% (95% CI: 95.8-99.0), PPV 84.1% (95% CI: 70.6-92.1), NPV 98.5% (95% IC: 96.6-99.4), Cohen´s kappa coefficient 0.80 (95% CI: 0.70-0.90). Conclusions: The test performed well, being better for P. vivax as compared to P. falciparum. There are still difficulties of RDT to detect low parasitemias. The non amplification of Pfhrp2 and Pfhrp3 genes in two samples diagnosed as mixed infection, suggest a possible deletion of these two genes together.

Seroprevalence and risk factors for cattle anaplasmosis, babesiosis, and trypanosomiasis in a Brazilian semiarid region / Soroprevalencia e fatores de risco para anaplasmose, babesiose e tripanosomiase bovina em uma região semiárida do Brasil

The seroprevalence of Anaplasma marginale, Babesia bigemina, Babesia bovis and Trypanosoma vivax and the risk factors for these infections were investigated in 509 cows on 37 farms in the semiarid region of Paraíba, northeastern Brazil. Cow serum samples were tested by means of immunofluorescence assay (IFA) against each specific antigen. The mean seroprevalence values per farm were 15.0% (range: 0-75%) for A. marginale, 9.5% (range: 0-40%) for B. bigemina and 26.9% (range: 0-73.7%) for B. bovis. All cows tested negative for T. vivax. Higher prevalence for A. marginale was significantly associated with less frequent acaricide spraying per year and with higher use of injectable antihelminthics. Presence of cows positive for B. bigemina was significantly associated with acaricide use and with presence of horse flies on the farm. Both occurrence and higher prevalence of B. bovis were significantly associated with recent observations of ticks on cattle. Overall, the present results indicate that the region investigated is an enzootically unstable area for A. marginale, B. bigemina and B. bovis, since most animals were seronegative to at least one agent.

A soroprevalência de Anaplasma marginale, Babesia bigemina, Babesia bovis e Trypanosoma vivax, assim como os fatores de risco para estas infecções, foram investigadas em 37 fazendas (total de 509 vacas) da região semiárida da Paraíba, nordeste do Brasil. A presença de anticorpos nos soros dos animais foi detectada pela técnica de imunofluorescência indireta, utilizando antígenos específicos. Os valores médios de soroprevalência por fazenda foram 15,0% (0-75%) para A. marginale, 9,5% (0-40%) para B. bigemina, e 26,9% (0-73,7%) para B. bovis. Todas as vacas foram soronegativas para T. vivax. As maiores prevalências de A. marginale foram significativamente associadas com menor uso de carrapaticidas por ano e com uso mais frequente de antihelmínticos injetáveis. A soroprevalência de B. bigemina foi significativamente associada com o uso de carrapaticidas, e com a presença de mutucas na fazenda. Tanto a ocorrência como a maior soroprevalência para B. bovis nas fazendas foram significativamente associadas com a presença recente de carrapatos nos bovinos. No geral, os resultados indicam que as fazendas amostradas estão situadas em área de instabilidade enzoótica para A. marginale, B. bigemina, e B. bovis, uma vez que a maioria dos animais foi soronegativa para pelo menos um dos agentes.

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil / Pesquisa sorológica por doenças transmitidas por carrapatos em cães da Amazônia oriental, Brasil

Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.

Ehrliquiose canina e babesiose canina são as doenças parasitárias transmitidas por carrapatos de maior prevalência em cães do Brasil. Poucos estudos pesquisaram doenças transmitidas por carrapatos na região da Amazônia brasileira. Um total de 129 amostras de sangue foram colhidas de cães da Amazônia oriental brasileira. Setenta e dois cães eram de áreas rurais de 19 municípios do Estado do Pará, e 57 amostras foram colhidas de cães errantes vadios da área urbana do município de Santarém-PA. As amostras de soro foram submetidas ao ensaio de imunofluorescência indireta, com antígenos de Babesia canis vogeli, Ehrlichia canis, e seis espécies de Rickettsia. A frequência de cães com anticorpos anti-B. canis vogeli, anti-E. canis, e anti-Rickettsia spp. foi de 42,6%, 16,2% e 31,7%, respectivamente. Anticorpos anti-B. canis vogeli foram detectados em 59,6% dos cães urbanos, e em 29,1% dos cães rurais (P < 0.05). Para E. canis, a soroprevalência foi parecida entre os cães urbanos (15,7%) e rurais (16,6%). Para Rickettsia spp., cães rurais apresentaram prevalência (P < 0.05) significativamente maior (40,3%) do que os cães urbanos (21,1%). Esse primeiro estudo sobre agentes transmitidos por carrapatos entre cães da Amazônia oriental brasileira indica que estes animais estão expostos a vários agentes. Estes incluem Babesia principalmente na área urbana, Riquétsias do grupo da Febre Maculosa principalmente nas áreas rurais, e Erliquia em cães de ambas as áreas, rural e urbana.

Seroprevalencia de la infección por Trypanosoma cruzi en Canis familiaris del estado Sucre, Venezuela / Seroprevalence of T. cruzi infection in Canis familiaris, state of Sucre, Venezuela.

Introducción. En Venezuela, la infección por T. cruzi en humanos ha sido ampliamente estudiada; sin embargo, en reservorios ha sido menos abordada. El objetivo de este trabajo fue determinar la seroepidemiología de la infección por T. cruzi en perros en el estado Sucre, Venezuela. Materiales y métodos. Se llevó a cabo un estudio prospectivo transversal en 95 centros poblados y 577 viviendas de los 15 municipios del estado Sucre, Venezuela, entre agosto y noviembre de 2008. Los sueros se evaluaron con el estuche CruziELISA y la prueba de unión de múltiples antígenos (Multiple Antigen Binding Assay, MABA). Además, se aplicaron encuestas epidemiológicas para evaluar los factores de riesgo. Resultados. Se evaluaron 363 perros (edad promedio: 2,6 ± 2,2 años, 226 machos y 137 hembras). Con la combinación de las pruebas ELISA/MABA se detectaron 78 sueros positivos, 69 negativos y 10 resultados inconclusos. La seroprevalencia de la infección para T. cruzi en perros, en el estado Sucre fue de 22,1 % (IC95%: 20,58-22,4). No se encontró asociación estadística significativa entre la infección por T. cruzi en perros y las variables epidemiológicas evaluadas: perros cazadores, dormir al aire libre, deambular libremente por el centro poblado, sexo del animal y hábitos de alimentación. La infección por T. cruzi estuvo asociada a la edad de los perros, y fue significativamente mayor en el grupo de 0 a 3 años, en comparación con los perros mayores. Conclusiones. La alta seroprevalencia detectada en los perros muestra que en esta región de Venezuela existe un factor de riesgo importante de transmisión de este parásito a poblaciones humanas.

Introduction: Trypanosoma cruzi infection in humans has been extensively studied in Venezuela; however, in reservoirs it has been less investigated. Objective: The objective of this study was to determine the seroepidemiology of T. cruzi in the state of Sucre, Venezuela. Materials and methods: A cross-sectional and prospective study conducted in 95 towns and 577 dwellings in the 15 municipalies of the state of Sucre, Venezuela, from August to November, 2008. The evaluation of serum samples was performed with the CruziELISA kit and the multiple antigens binding assays (MABA). Furthermore, epidemiological surveys were applied to evaluate the risk factors. Results: A total of dogs (average age of 2, 6 + 2.2 years, 226 males and 137 females) was evaluated. The combination of the ELISA / MABA tests detected 78 positive sera, sixty-nine negative and 10 of inconclusive results. The seroprevalence of the T. cruzi infection in dogs in the state of Sucre, was 22.1% (CI 95%: 20.58-22.4%). No significant statistic association was found between the T. cruzi infection in dogs and the evaluated epidemiological variables: hunting dogs that slept oudoors roaming freely in the populated center, sex of the animal and eating habits. The T. cruzi infection was associated to the age of canines, being significantly higher in the group of 0 to 3 years, when compared with older dogs. Conclusions: The high T. cruzi seroprevalence dected in dogs shows that in this región of Venezuela there prevails an important risk factor of transmissibility of this parasite to human populations.

Recent advances in the diagnosis and treatment of kala-azar.

In India, about 100 000 cases of visceral leishmaniasis (VL) or kala-azar are estimated to occur annually, 90% of which occur in the state of Bihar. Currently, antibody-based tests such as the rK39-based immunochromatographic strip test and the direct agglutination test (DAT) are widely used for the diagnosis of VL. However, their major drawback is continued positivity both long after cure and in a high proportion of individuals living in endemic areas. Thus, antibody-based tests must always be used in combination with a standardized clinical case definition for VL. There have been many breakthroughs in the past decade in the treatment of kala-azar in India, such as approval of oral miltefosine and paromomycin, single-dose treatment with liposomal amphotericin B and multidrug treatment. Encouraged by these advances, an ambitious VL elimination programme was launched with the aim to eliminate VL as a public health problem in India, Nepal and Bangladesh by 2015. Early diagnosis, complete treatment of cases, integrated vector management, effective disease surveillance, and clinical and operational research should be the five key components of the strategy to achieve this goal.

A Recombinant Plasmodium vivax Apical Membrane Antigen-1 to Detect Human Infection in Iran

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.

Malaria seroprevalence in blood bank donors from endemic and non-endemic areas of Venezuela

In Venezuela, a total of 363,466 malaria cases were reported between 1999-2009. Several states are experiencing malaria epidemics, increasing the risk of vector and possibly transfusion transmission. We investigated the risk of transfusion transmission in blood banks from endemic and non-endemic areas of Venezuela by examining blood donations for evidence of malaria infection. For this, commercial kits were used to detect both malaria-specific antibodies (all species) and malaria antigen (Plasmodium falciparum only) in samples from Venezuelan blood donors (n = 762). All samples were further studied by microscopy and polymerase chain reaction (PCR). The antibody results showed that P. falciparum-infected patients had a lower sample/cut-off ratio than Plasmodium vivax-infected patients. Conversely, a higher ratio for antigen was observed among all P. falciparum-infected individuals. Sensitivity and specificity were higher for malarial antigens (100 and 99.8 percent) than for antibodies (82.2 and 97.4 percent). Antibody-positive donors were observed in Caracas, Ciudad Bolívar, Puerto Ayacucho and Cumaná, with prevalences of 1.02, 1.60, 3.23 and 3.63 percent, respectively. No PCR-positive samples were observed among the donors. However, our results show significant levels of seropositivity in blood donors, suggesting that more effective measures are required to ensure that transfusion transmission does not occur.

Evaluation of Rapid Diagnostics for Plasmodium falciparum and P. vivax in Mae Sot Malaria Endemic Area, Thailand

Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pantrade mark, Malaria Ag-Pftrade mark, and Malaria Ag-Pvtrade mark tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pftrade mark and Malaria Antigen Pf/Pantrade mark compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, and Malaria Antigen Pf/Pantrade mark were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pftrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.

Antigenic characterization of Trypanosoma evansi using sera from experimentally and naturally infected bovines, equines, dogs, and coatis / Caracterização antigênica do Trypanosoma evansi usando soros de bovinos, equinos, cães e quatis experimentalmente e naturalmente infectados

The present research investigated the presence of T. evansi antibodies in animals from the subregion of Nhecolandia, in the Pantanal Sul-mato-grossense, by means of an enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT), and the pattern of polypeptide recognition by sera from experimentally and naturally infected hosts using Western blotting. Serum samples were obtained from bovines (n = 102), horses (n = 98), and dogs (n = 55), and from 32 free-ranging coatis (Nasua nasua). None of the bovines were found positive, while sera from 16 dogs (29 percent) and 23 horses (23.4 percent) were positive by ELISA. Sera from 8 coatis (25 percent) were found positive using IFAT. Western blotting revealed major polypeptides of T. evansi with molecular weight ranging from 74 to 38 kDa. The polypeptides of 66, 48-46, and 38 kDa were identified by sera from experimentally infected bovines, donkeys, dogs, and coatis. The 48-46 and 38 kDa bands were mainly recognized in chronic phase of infection. The antigen with apparent molecular weight of 66 kDa, revealed by antibodies from all experimental animals, was also recognized in sera of horses and dogs from the Pantanal. The 48-46 kDa polypeptide was identified by antibodies from all naturally infected animals and must be further evaluated for use in specific diagnosis of T. evansi infection.

O trabalho de pesquisa investigou a presença de anticorpos anti- T. evansi em animais da sub-região da Nhecolândia, no Pantanal sul-mato-grossense, pelo ensaio imunoenzimático (ELISA) e a reação de imunofluorescência indireta (RIFI). O reconhecimento de polipeptídeos de T. evansi foi realizado pela técnica de "Western blotting", utilizando soros de animais experimentalmente e naturalmente infectados. As amostras de soro foram obtidas de bovinos (n = 102), cavalos (n = 98) e cães (n = 55) e de 32 quatis de vida livre (Nasua nasua) do pantanal mato-grossense. Todos os soros dos bovinos foram negativos, enquanto soros de 16 cães (29 por cento) e 23 cavalos (23,4 por cento) foram positivos pelo ELISA. Soros de oito quatis (25 por cento) foram positivos pela RIFI. Pelo "Western-blotting" foi possível revelar polipeptídeos de T. evansi, com peso molecular variando de 74 a 38 kDa. Os polipeptídeos de 66, 48-46 e 38 kDa foram identificados por soros de bovinos, cavalos, cães e quatis experimentalmente infectados. As bandas de 48-46 e 38 kDa foram reconhecidas principalmente na fase crônica da infecção. O antígeno com peso molecular aparente de 66 kDa, revelado por anticorpos de todos os animais experimentais, também foi reconhecido por soros de cavalos e cães do Pantanal. O polipeptídeo de 48-46 kDa foi identificado por anticorpos de todos os animais naturalmente infectados, devendo ser avaliado para o diagnóstico específico da infecção pelo T. evansi.

Congenital toxoplasmosis transmitted by human immunodeficiency-virus infected women

We report the occurrence of congenital toxoplasmosis in three infants born to HIV infected women who had high anti-toxoplasma IgG and negative IgM during pregnancy. We briefly reviewed available literature and discussed the possible transmission mechanisms of congenital toxoplasmosis among HIV infected pregnant women. Serum samples were tested for Toxoplasma gondii IgM and IgG antibodies using commercial enzyme immunoassay and IgG-avidity tests. In the first case, fetal death occurred at 28th week of gestation. In the second case, congenital toxoplasmosis was diagnosis at 6th month of life; and in the third case, an HIV-infected newborn, congenital toxoplasmosis was asymptomatic. These cases point out to the possibility of enhanced maternal-fetal transmission of T. gondii infection by HIV-infected women chronically infected, which may have important public health consequences, considering that increasing frequency of HIV-infection has been observed among women of childbearing age around the world.