Serological detection and risk factors for equine piroplasmosis in the semiarid region of Pernambuco, Northeastern Brazil / Detecção sorológica e fatores de riscos para piroplasmose equina na região semiárida de Pernambuco, Nordeste do Brasil

Abstract Equine piroplasmosis, an economically important disease in horses, has so far not been reported in Pernambuco state, Brazil. This study aimed to evaluate the seroprevalence of anti-Babesia caballi and anti-Theileria equi antibodies based on the detection of these agents in equine blood and in ticks on horses in the municipality of Petrolina, Pernambuco, northeastern Brazil. Blood samples were drawn from 393 horses and sera were examined by ELISA. The presence of tick infestations was evaluated, and 101 ticks were subjected to DNA amplification for the detection of Babesia spp. by polymerase chain reaction (PCR). No parasites were detected in the blood smears. Anti-B. caballi and anti-T. equi antibodies were found in 27.2% (107/393) and 34.8% (137/393) horses, respectively. Infestation by Dermacentor nitens was detected in 4.3% (17/393) of the horses. There was no DNA amplification of the agents in ticks. The risk factors for the presence of anti-T. equi antibodies (P < 0.05) were: purebred (P < 0.001), animals older than 156 months (P = 0.014), and the presence of ticks (P = 0.001). No risk factors for B. caballi were identified. This study confirmed the circulation of agents of equine piroplasmosis in the municipality of Petrolina, state of Pernambuco, Brazil.

Resumo Piroplasmose equina é uma doença economicamente importante em equinos e não possui relatos no Estado de Pernambuco, Brasil. O objetivo deste estudo foi avaliar a soroprevalência de anticorpos anti-B. caballi e anti-T. equi pela detecção destes agentes no sangue e carrapatos de equinos no município de Petrolina, Pernambuco, Nordeste do Brasil. Amostras de sangue de 393 equinos foram coletadas e submetidas ao esfregaço sanguíneo e ELISA. A presença de infestação por carrapatos foi avaliada, e 71 carrapatos foram submetidos à Reação em Cadeia da Polimerase (PCR) para Babesia spp. Nenhum parasito foi detectado na análise de esfregaços de sangue. Anticorpos anti-B. caballi e anti-T. equi foram verificados em 27,2% (107/393) e 34,8% (137/393) dos equinos, respectivamente. A infestação por Dermacentor nitens foi verificada em 4,3% (17/393) dos equinos. Não houve amplificação do DNA dos agentes nos 71 carrapatos submetidos à PCR. Os fatores de risco para presença de anticorpos anti-T. equi (P < 0,05) foram: raça definida (P < 0,001), animais > de 156 meses (P = 0,014) e presença de carrapatos (P = 0,001). Nenhum fator de risco foi identificado para B. caballi. Esse estudo permitiu a confirmação da presença de agentes da piroplasmose equina no município de Petrolina, Pernambuco.

Seroprevalencia y detección molecular de Toxoplasma gondii en donantes de un banco de sangre de Cúcuta, Colombia / Seroprevalence and molecular detection of Toxoplasma gondii among donors in a blood bank in Cúcuta, Colombia

Resumen Introducción. La infección por Toxoplasma gondii puede presentarse en los humanos con un amplio rango de manifestaciones que van desde el estado asintomático hasta la enfermedad grave, según el estado inmunológico del individuo. Los mecanismos de transmisión incluyen la transfusión sanguínea, pero poco se sabe sobre la frecuencia del parásito en los bancos de sangre de Colombia. Objetivo. Determinar la prevalencia de la infección con T. gondii en donantes de un banco de sangre de Cúcuta mediante técnicas de diagnóstico serológico y molecular. Materiales y métodos. Se determinaron los anticuerpos IgG e IgM contra T. gondii mediante un inmunoensayo en suero en 348 donantes. Se determinó la frecuencia de ADN de T. gondii utilizando la reacción en cadena de la polimerasa (PCR) en sangre total de donantes seropositivos y se analizaron las variables de interés con base en la información obtenida durante la selección de donantes. Resultados. De los 348 donantes participantes, 134 (38,5 %) presentaron anticuerpos IgG contra T. gondii; dos (0,6 %) de ellos presentaron tanto IgG como IgM y, en dos (1,5 %), se detectó ADN del parásito en la sangre. Un análisis bivariado evidenció una asociación entre la seropositividad para T. gondii y tener más de 26 años de edad (p=0,020). Conclusiones. La prevalencia de la infección con T. gondii encontrada en los donantes de sangre sugiere una exposición significativa al agente, la cual adquiere relevancia al detectarse la parasitemia.

Abstract Introduction: Toxoplasma gondii infection manifests differently in humans according to their immunity ranging from asymptomatic profiles to severe disease. There are multiple transmission mechanisms including blood transfusions, but little is known about the frequency of T. gondii infection in Colombia's blood banks. Objective: To determine the prevalence of T. gondii infection in blood donors of a blood bank in the city of Cúcuta by serological and molecular diagnostic techniques. Materials and methods: We identified IgG and IgM antibodies against T. gondii by immunoassay in serum from 348 donors. The frequency of T. gondii DNA was determined by polymerase chain reaction (PCR) in whole blood from seropositive donors and relevant variables were analyzed based on the information obtained from surveys during blood donor selection. Results: Out of the 348 enrolled donors, 134 (38.5%) showed IgG antibodies against T. gondii; two of them (0.6%) had both IgG and IgM, and in two of them (1.5%), parasite DNA was detected in blood samples. A bivariate analysis indicated an association between seropositivity to T. gondii and being over 26 years of age (p=0.020). Conclusions: The prevalence of T. gondii infection found in the blood donors of this study suggests a significant exposure to the infectious agent that becomes relevant when parasitemia is detected.

Toxoplasma gondii and Neospora caninum infections in synanthropic rodents from Argentina / Infecções por Toxoplasma gondii e Neospora caninum em roedores sinantrópicos da Argentina

Abstract Toxoplasma gondii and Neospora caninum are closely related coccidian parasites (phylum Apicomplexa). This is the first study from urban synanthropic rodent species that involved serological and molecular diagnosis of T. gondii and N. caninum infection, and genotyping of T. gondii in Argentina. A total of 127 rodent samples were trap captured: Mus musculus (n = 78), Rattus norvegicus (n = 26) and Rattus rattus (n = 23). Antibodies against T. gondii and N. caninum were detected by IFAT in 32.8% (40/122) and 0.8% (1/122) of rodent samples, respectively, demonstrating contact with these protozoans. Additionally, T. gondii DNA was detected in 3.3% (4/123) of rodent central nervous system samples and 2 samples were genotyped by multilocus nPCR-RFLP. Neospora caninum DNA was not detected by PCR. The 2 genotyped samples were type III allele for all markers except for SAG-1 (type I for Rat1Arg and type II/III for Rat2Arg) and were identified as #48 and #2 (likely) according to the allele combinations reported on Toxo DB (Toxo-DB). The results of the present study revealed a wide distribution of T. gondii and less for N. caninum, in synanthropic rats and mice in the studied area.

Resumo Toxoplasma gondii e Neospora caninum são parasitas coccídeos intimamente relacionados (filo Apicomplexa). Este é o primeiro estudo de espécies de roedores sinantrópicos urbanos, o qual envolveu diagnósticos sorológicos e moleculares da infecção por T. gondii e N. caninum e genotipagem de T. gondii na Argentina. Um total de 127 amostras de roedores foram obtidas: Mus musculus (n = 78), Rattus norvegicus (n = 26) e Rattus rattus (n = 23). Anticorpos contra T. gondii e N. caninum foram detectados pela IFAT em 32,8% (40/122) e 0,8% (1/122) das amostras de roedores, respectivamente, demonstrando contato com esses protozoários. Adicionalmente, o DNA de T. gondii foi detectado em 3,3% (4/123) das amostras do sistema nervoso central de roedores e duas amostras foram genotipadas por nPCR-RFLP multilocus. O DNA de N. caninum não foi detectado por PCR. As 2 amostras genotipadas eram do tipo III para todos os marcadores, exceto para SAG-1 (tipo I para Rat1Arg e tipo II / III para Rat2Arg) e foram identificadas como # 48 e # 2 (provavelmente) de acordo com as combinações de alelos relatadas no Toxo DB (Toxo-DB). Os resultados do presente estudo indicam uma ampla distribuição de T. gondii e menor para N. caninum , em ratos e camundongos sinantrópicos na área estudada.

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates / Técnicas sorológicas e moleculares aplicadas na identificação de Plasmodium spp. em amostras de primatas não humanos

Abstract The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

Resumo Neste estudo objetivamos identificar Plasmodium spp. em amostras sangue de primatas não humanos (PNH) do estado do Maranhão, utilizando técnicas clássicas e alternativas para o exame da malária humana. Foram analisadas 161 amostras de sangue de PNH, sendo 141 de CETAS (cativeiro) e 20 de reserva particular (vida livre), utilizando microscopia, teste de diagnóstico rápido (RDT), imunofluorescência indireta (IFI) e técnicas moleculares (semi-nested PCR, PCR em tempo real quantitativo e LAMP). Dois métodos sorológicos (dot-ELISA e ELISA indireto) também foram padronizados com antígenos solúveis de roptrias de P. falciparum e P. berghei. Formas trofozoíticas de Plasmodium sp. foram identificadas em lâminas de cinco animais diferentes. Nenhuma amostra foi positiva em TDR e LAMP. Quatro amostras foram soropositivas para P. malariae na IFI. Os soros de PNH mostraram baixa reatividade pelo ELISA indireto. Plasmodium sp. foi detectado em 34,16% (55/161) das amostras utilizando a qPCR baseada no gene 18S rRNA. No sequenciamento, duas amostras mostraram identidade com P. malariae (100%), uma com Plasmodium sp. ZOOBH (97%) e uma com P. falciparum (99%). A PCR mostrou ser a técnica mais sensível para diagnósticos de Plasmodium em amostras de PNH.

Comparison of optical microscopy and quantitative polymerase chain reaction for estimating parasitaemia in patients with kala-azar and modelling infectiousness to the vector Lutzomyia longipalpis

Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors.

Occurrence of anti-Toxoplasma gondii antibodies and parasite DNA in backyard chicken breeding in Northeast, Brazil / Ocorrência de anticorpos anti-Toxoplasma gondii e DNA do parasita em galinhas de criações domésticas no Nordeste, Brasil

Abstract The aim of the present study was to investigate the occurrence of anti-Toxoplasma gondii antibodies and parasite DNA in backyard chickens bred in the metropolitan area of Recife, Brazil. In total, 212 serum samples were collected from 16 properties, and 12 backyard chickens were collected in the six sanitary districts of Recife. An indirect immunofluorescence assay (IFA) was used to investigate the occurrence of anti-Toxoplasma gondii antibodies. Polymerase chain reaction (PCR) was used to detect T. gondii DNA in brain, heart, liver and lung specimens. Of the samples analyzed by serology, 86/212 (40.56%) were positive; of the samples analyzed by PCR, 2/12 (16.7%) were positive, with both samples positive by both tests (serological and molecular). The presence of antibody anti-T. gondii and parasite DNA in tissues of these animals are worrying aspects for public health because there is a risk of transmission of the parasite to humans through eating undercooked or raw meat. Based on the results, the adoption of preventive measures to prevent the cats access to the chickens creations should be encouraged, since these animals were identified in most of the studied properties.

Resumo O objetivo do presente estudo foi investigar a ocorrência de anticorpos anti-Toxoplasma gondii e de DNA do parasito em galinhas de criações domésticas, na região metropolitana de Recife, Brasil. No total, 212 amostras de soro foram coletadas de aves de 16 estabelecimentos e de 12 galinhas de criações domésticas nos seis distritos sanitários de Recife. Para a pesquisa de anticorpos anti-Toxoplasma gondii foi utilizada a Reação de Imunofluorescência Indireta (RIFI). A Reação em Cadeia da Polimerase (PCR) foi utilizada para detectar o DNA de T. gondii em fragmentos de cérebro, coração, fígado e pulmão. Das amostras analisadas por sorologia, 86/212 (40,56%) foram positivas. Das amostras analisadas por PCR, 2/212 (16,7%) foram positivas, em ambos os testes (sorológicos e moleculares). A presença de anticorpos anti-T. gondii e de DNA parasitário nos tecidos desses animais são aspectos preocupantes para saúde pública, porque há o risco de transmissão do parasita para humanos através da ingestão de carne mal cozida ou crua. Com base nos resultados obtidos, a adoção de medidas preventivas que evitem o acesso de gatos às criações de galinhas deve ser incentivada, uma vez que esses animais foram identificados na maioria das propriedades estudadas.

Detection of Toxoplasma gondii DNA in peripheral blood and aqueous humor of patients with Toxoplasmic active focal necrotizing retinochoroiditis using real-time PCR / Detecção de DNA do Toxoplasma gondii no sangue periférico e humor aquoso por PCR em tempo real

ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR) for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43) of the aqueous humor samples and 2.33% (1/43) of the peripheral blood samples; further, 16.27% (7/43) of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

RESUMO Objetivo: Analisar o uso do PCR em tempo real (qPCR) na detecção do DNA do T. gondii no sangue periférico e no humor aquoso de pacientes com lesões de retinocoroidite focal, ativa por toxoplasmose. Métodos: Cinquenta e cinco pacientes com uveite infecciosa foram incluídos neste estudo. Os pacientes foram atendidos entre 2009 a 2013, no Departamento de Oftalmologia e Ciências Visuais da Universidade Federal de São Paulo. Quarenta e três pacientes tiveram o diagnóstico de lesões de retinocoroidite focal, ativa por toxoplasmose e, os outros 12 tiveram o diagnóstico de uveíte infecciosa não toxoplásmica e, por isso foram usados como grupo controle. A técnica de qPCR foi utilizada na detecção de DNA do T. gondii em amostras de sangue periférico e humor aquoso. Resultados: O qPCR foi positivo para o DNA do T. gondii em 37,21% (16/43) das amostras de humor aquoso, 2,33% (1/43) nas amostras de sangue periférico e, 16,27% (7/43) em ambas amostras simultaneamente. Conclusão: O qPCR foi capaz de detectar o DNA do T. gondii em pacientes com lesões de retinocoroidite focal, ativa por Toxoplasmose, no sangue bem como, no humor aquoso, podendo ajudar no diagnostico.

Seropositivity and Serointensity of Toxoplasma gondii Antibodies and DNA among Patients with Schizophrenia

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.

Seropositivity and Serointensity of Toxoplasma gondii Antibodies and DNA among Patients with Schizophrenia

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.

Evaluación preliminar de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® en individuos colombianos / Preliminary evaluation of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® in Colombian individuals

Introducción. El diagnóstico de la enfermedad de Chagas es fundamental para brindar un tratamiento oportuno y mejorar el pronóstico del paciente. La capacidad discriminatoria de las pruebas serológicas para el diagnóstico varía de acuerdo con la prevalencia de la enfermedad y el antígeno utilizado en la prueba. Objetivo. Evaluar la capacidad discriminatoria de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (NovaTec Immunodiagnostica GmbH) en un grupo de individuos colombianos utilizando la prueba de inmunofluorescencia indirecta (IFI) y el ensayo de inmunoabsorción enzimática (ELISA) como referencia. Materiales y métodos. Se incluyeron 78 muestras de pacientes crónicos (36 asintomáticos y 42 sintomáticos) y 21 de controles sanos. También se analizaron 17 individuos no infectados con riesgo epidemiológico para la enfermedad de Chagas, siete con leishmaniasis y nueve con enfermedad cardiaca. Se evaluaron por PCR en tiempo real cuatro individuos cuyos resultados variaron entre pruebas. Resultados. Se encontraron diferencias significativas a una densidad óptica de 450 nm (p<0,0001) al comparar la mediana de la absorbancia entre los controles sanos (0,143) y los asintomáticos (2,401) o sintomáticos (2,776), entre los asintomáticos y sintomáticos (p=0,0408), entre los seronegativos con riesgo (0,232), individuos con enfermedades cardiacas (0,367) o con leishmaniasis (0,337) y los pacientes con enfermedad de Chagas (p<0,0001), y entre los controles sanos y los pacientes seronegativos con riesgo (p=0,0264), con enfermedades cardiacas (p=0,0015) o con leishmaniasis (p=0,002). La PCR en tiempo real fue positiva en tres de los cuatro casos. Conclusiones. Esta prueba comercial de ELISA permitió discriminar a los pacientes con Chagas de los controles. Se requieren estudios de fase II para determinar las características operativas de la prueba.

Introduction: The diagnosis of Chagas´ disease is essential to provide early treatment and improve patients´ prognosis . The discriminatory efficiency of the serological tests varies according to the disease prevalence and the test- antigen used . Objective: To evaluate the discriminatory efficiency of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (Nova Tec Immunodiagnostica GmbBH) in a group of Colombian individuals, using indirect immunofluorescence antibody testing (IFAT) and enzyme immunoassay (ELISA) tests as references. Materials and methods: Seventy-eight samples from chronic chagasic patients (36 asymptomatic and 42 symptomatic) and 21 healthy controls were included. Seventeen samples from non-infected people with Chagas´ disease epidemiological risk, seven with leishmaniasis and nine with non-chagasic cardiomyopathy were also analyzed. Real time PCR was performed on four individuals whose results differed among tests. Results: Significant differences at 450 nm optical absorbance were found (p<0.0001) when the median absorbance values of healthy controls (0.143), asymptomatic (2.401) and symptomatic (2.776) chagasic patients were compared, as well as when asymptomatic and symptomatic patients (p=0.0408) and seronegative people with epidemiological risk (0.232), cardiomyopathy (0.367) or leishmaniasis (0.337) were compared with chagasic patients (p<0.0001). Finally, there were differences among healthy controls and non-infected people with epidemiological risk (p=0.0264), patients with non-chagasic cardiomyopathy (p=0.0015) and patients with leishmaniasis (p=0.002). Real-time PCR was positive in three out of four analyzed cases. Conclusions: The commercial ELISA test allowed us to discriminate the chagasic patients from the controls. A phase II study of diagnostic tests for determining field reliability of this test is required.

Estudio retrospectivo de la aplicación de la amplificación génica cualitativa en muestras biológicas para el seguimiento de la toxoplasmosis en pacientes pediátricos receptores de trasplante de células progenitoras hematopoyéticas / Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantion

La toxoplasmosis es una infección oportunista causada por el parásito Toxoplasma gondii; su infección es grave y de difícil diagnóstico en pacientes que reciben un trasplante alogénico de células progenitoras hematopoyéticas (TCPH). En el Hospital de Pediatría S.A.M.I.C. "Profesor Dr. Juan P. Garrahan" se realizó la vigilancia postrasplante de 12 pacientes receptores de TCPH mediante la técnica de PCR cualitativa. La necesidad de seguimiento de estos pacientes fue definida por el antecedente de serología positiva para toxoplasmosis en el donante o receptor y ante la imposibilidad de iniciar el uso profiláctico de trimetoprima-sulfametoxazol a causa de la condición hematológica. Dos pacientes presentaron signos de enfermedad por T. gondii con resultado de PCR positivo y recibieron tratamiento con pirimetamina-clindamicina. En otros dos, la toxoplasmosis fue causa de muerte y hallazgo de autopsia, con resultado de PCR negativo. Cuatro pacientes recibieron tratamiento contra toxoplasmosis por la detección de una PCR positiva, sin manifestaciones clínicas. En los cuatro pacientes restantes no se detectaron signos de enfermedad por toxoplasmosis, con resultados de PCR negativos durante el seguimiento. La técnica de PCR cualitativa demostró ser útil para detectar la reactivación de la toxoplasmosis en receptores de TCPH, pero tiene limitaciones para el seguimiento y la toma de decisiones clínicas en pacientes con PCR positiva que persiste en el tiempo y manifestaciones de toxicidad por el tratamiento.

Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment.

First Evaluation of an Outbreak of Bovine Babesiosis and Anaplasmosis in Southern Brazil Using Multiplex PCR

Outbreaks of tick-borne disease cases in Santa Catarina, Brazil are known, but the presence of the pathogen DNA has never been determined. In this study, the first survey of Anaplasma marginale, Babesia bigemina, and Babesia bovis DNA on blood samples of 33 cattle from an outbreak in Ponte Alta Municipality, Santa Catarina, Brazil, has been carried out. A multiplex PCR detected 54.5% of animals were co-infected with 2 or 3 parasites, while 24.2% were infected with only 1 species. The most prevalent agent was B. bigemina (63.6%) followed by A. marginale (60.6%). This is the first report of tick-borne disease pathogens obtained by DNA analysis in Southern Brazil.

Molecular Detection and Seroprevalence of Babesia microti among Stock Farmers in Khutul City, Selenge Province, Mongolia

Babesiosis is an emerging tick-borne disease in humans worldwide; however, little is known about the frequency of infection or prevalence of this disease in other parts of the world, excluding North America. In this study, we aimed to investigate Babesia microti infection frequency in a human population in Mongolia. One hundred blood samples were collected from stock farmers living in Khutul city of Selenge province, Mongolia. The sera and DNA from blood samples were evaluated for the presence of B. microti infection by using indirect fluorescent antibody (IFA) tests and PCR. The positive detection rates obtained using the IFA tests and PCR assays were 7% and 3%, respectively. This study is the first to detect of B. microti infections based on antibody seroprevalence or PCR assays for the presence of B. microti DNA in a Mongolian population.

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

First description of autochthonous canine visceral leishmaniasis in the metropolitan region of Vitória, State of Espírito Santo, Brazil / Primeira descrição de leishmaniose visceral canina autóctone na Região Metropolitana de Vitória, Estado do Espírito Santo, Brasil

INTRODUCTION: We investigated autochthonous canine visceral leishmaniasis (CVL) in the metropolitan region of Vitória (MRV), an area in which a human case was previously reported. METHODS: Serological, parasitological, and molecular tests were performed in 201 dogs. RESULTS: Twenty-six (13%) and 12 (6%) dogs were identified as positive using in-house enzyme-linked immunosorbent assay (ELISA) and rK39 tests, respectively. Two dogs had a positive culture for Leishmania chagasi, and 4 were polymerase chain reaction (PCR)-positive for Leishmania spp. One positive dog belonged to the aforementioned patient. CONCLUSIONS: Although the responsible vector was not found, our results provide evidence of autochthonous CVL in the MRV, a non-endemic area for VL.

INTRODUÇÃO: Descrevemos um foco de leishmaniose visceral canina (LVC) autóctone na Região Metropolitana de Vitória (RMV) onde um caso humano foi registrado anteriormente. MÉTODOS: Testes sorológicos, parasitológicos e moleculares foram realizados em 201 cães. RESULTADOS: Vinte e seis (13%) e 12 (6%) foram positivos para um teste ELISA in house e rK39, respectivamente. Dois cães apresentaram cultura positiva para Leishmania (Leishmania) chagasi e quatro PCR positivo para Leishmania spp. Um dos cães positivo pertencia ao paciente supracitado. CONCLUSÕES: Embora o vetor não tenha sido encontrado, nossos resultados fornecem evidências da LVC autóctone na RMV, área não-endêmica para leishmaniose visceral.

Seroepidemiología del paludismo en un grupo de migrantes en tránsito (Chiapas, 2008)

OBJETIVO: Conocer la prevalencia del paludismo y los factores asociados con la infección de migrantes en la frontera sur de México, durante 2008. MATERIAL Y MÉTODOS: En 706 migrantes, se investigó la infección activa mediante prueba rápida y PCR o pasada, mediante serología y se aplicó un cuestionario para investigar las condiciones asociadas con la infección. RESULTADOS: 85.6% provenía de Centroamérica. Ninguno presentó infección activa; 4.2% fue seropositivo y la mayoría provenía de los países con mayor incidencia de paludismo en la región. La seropositividad se asoció con el número de episodios previos de paludismo (RM=1.44; IC95% 1.04-2.00), años de permanencia en su comunidad de origen (RM=1.03; IC95% 1.00 -1.07) y conocimiento y automedicación con antipalúdicos (RM=3.38; IC95% 1.48-7.67). CONCLUSIONES: La exposición previa de migrantes al paludismo y las dificultades para su detección indican la necesidad de nuevas estrategias para la vigilancia epidemiológica para estas poblaciones.

OBJECTIVE: To know the prevalence of malaria and the factors associated with the infection in migrants in the southern border of Mexico, during 2008. MATERIALS AND METHODS: In 706 migrants, active malaria infection was investigated using a rapid diagnostic test and PCR and past infection using serology. A questionnaire was applied to investigate the conditions associated to infection. RESULTS: 85.6% originated from Central America, none presented an active infection, although 4.2% were seropositive, most of these came from the countries with the highest malaria incidence in the region. Seropositivity was associated with the number of previous malaria episodes (OR=1.44; IC95% 1.04-2.00), years living in their community of origin (OR=1.03; IC95% 1.00-1.07), and knowledge and self-medication with anti-malaria drugs (OR=3.38; IC95% 1.48-7.67). CONCLUSIONS:. The previous exposure of migrants and the difficulties for their detection indicate the need of new strategies for the epidemiological surveillance for these populations.

Desenvolvimento de sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral / Development systems based on nested-PCR and conventional single tube for the diagnosis of visceral leishmaniasis

A leishmaniose visceral ocorre em países dos cinco continentes e quando não tratada pode levar a óbito. Para se evitar esse desfecho, são essenciais o diagnóstico precoce e tratamento adequado. Com o objetivo de contribuir na pesquisa de novos diagnósticos para leishmaniose visceral, esse trabalho propôs desenvolver sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral. A partir de uma revisão na literatura em busca de alvos moleculares utilizados no diagnóstico dessa parasitose, foram selecionados os alvos subunidade menor do RNA ribossômico (ssu rRNA) e espaçador transcrito interno 1 (ITS-1), que compõem o DNA do agente etiológico Leishmania infantum, para o desenvolvimento das nested-PCR. Foi também escolhido o alvo kDNA, o mais aplicado nas abordagens de PCR, para comparações com os sistemas desenvolvidos. Após otimizar todas as PCR com DNA genômico de L. infantum, esses sistemas foram avaliados em amostras de sangue, soro e urina de indivíduos com suspeita de leishmaniose visceral dos hospitais de referência da cidade do Recife - PE. Para utilização da urina, foram avaliados quatro protocolos de extração de DNA e identificou-se que a extração por fenol-clorofórmio, com modificações, foi a de melhor desempenho. Na avaliação com amostras biológicas, as PCR simples e nested-PCR com os alvos ssu rRNA e ITS-1 não tiveram boa sensibilidade ao se usar sangue, e não foram capaz de amplificar DNA do parasito em soro e urina. Esses sistemas desenvolvidos não podem ser usados para o diagnóstico da leishmaniose visceral. No entanto, a kDNAPCR apresentou bons resultados quando avaliada com urina. Mais estudos devem ser feitos para avalia-la como um diagnóstico seguro para esse tipo de amostra biológica. Esse trabalho representa um ponto de início para posteriores estudos que objetivem o aprimoramento e validação da nested-PCR único tubo para o diagnóstico da leishmaniose visceral.

Serologic Survey of Toxoplasmosis in Seoul and Jeju-do, and a Brief Review of Its Seroprevalence in Korea

Knowledge of the prevalence of human Toxoplasma gondii infection is required in the Republic of Korea. In this study, we surveyed the seroprevalence of T. gondii infection and analyzed the risk factors associated with seropositivity among residents in 2 administrative districts; Seoul and the island of Jeju-do, which have contrasting epidemiologic characteristics. Sera and blood collected from 2,150 residents (1,114 in Seoul and 1,036 in Jeju-do) were checked for IgG antibody titers using ELISA and for the T. gondii B1 gene using PCR. In addition, participants completed a questionnaire that solicited information on gender, age, occupation, eating habits, history of contact with animals, and travel abroad. The T. gondii B1 gene was not detected in all residents examined. However, ELISA showed 8.0% (89 of 1,114 sera) positive for IgG antibodies against T. gondii in Seoul and 11.3% (117 of 1,036 sera) in Jeju-do. In both districts, the positive rates were higher in males than in females, and those 40-79 years of age showed higher rates than other ages. In Seoul, residents older than 70 years of age showed the highest positive rate, 14.9%, whereas in Jeju-do the highest prevalence, 15.6%, was in those in their sixties. The higher seropositive rate in Jeju-do than in Seoul may be related to eating habits and occupations. The present results and a review of related literature are indicative of an increased seroprevalence of T. gondii in Korea in recent years.

Importance of high IgG anti-Toxoplasma gondii titers and PCR detection of T. gondii DNA in peripheral blood samples for the diagnosis of AIDS-related cerebral toxoplasmosis: a case-control study

BACKGROUND: Cerebral toxoplasmosis (CT) continues to cause significant morbidity and mortality in human immunodeficiency virus (HIV)-infected patients in Brazil. In clinical practice, the initial diagnosis is usually presumptive and alternative diagnosis tools are necessary. Our objective was to evaluate whether the detection of high titers of IgG anti-Toxoplasma gondii and T. gondii DNA in blood samples are associated with the diagnosis of CT. METHODS: In this case-control study we included 192 patients with HIV-1 infection: 64 patients with presumptive CT (cases) and 128 patients with other diseases (controls). Blood samples to perform indirect immunofluorescense reaction (IFI) to detect anti-T. gondii IgG antibodies and polymerase chain reaction (PCR) were collected before or within the first three days of anti-Toxoplasma therapy. Two multivariate logistic regression models were performed: one including the variable qualitative serology and another including quantitative serology. RESULTS: In the first model, positive IgG anti-T. gondii (OR 4.7, 95 percent CI 1.2-18.3; p = 0.027) and a positive T. gondii PCR result (OR 132, 95 percent CI 35-505; p < 0.001) were associated with the diagnosis. In the second model, IgG anti-T. gondii titres > 1:1024 (OR 7.6, 95 percent CI 2.3-25.1; p = 0.001) and a positive T. gondii PCR result (OR 147, 95 percent CI 35-613; p < 0.001) were associated with the diagnosis. CONCLUSIONS: Quantitative serology and molecular diagnosis in peripheral blood samples were independently associated with the diagnosis of CT in HIV-infected patients. These diagnostic tools can contribute to a timely diagnosis of CT in settings where Toxoplasma infection is common in the general population.