Antibodies against Leptospira spp. in bovine serum samples from several Brazilian states analyzed in the period from 2007 to 2015 / [Anticorpos contra Leptospira spp. em amostra de soro bovino de diversos estados brasileiros analisados no período entre 2007 e 2015]

Bovine leptospirosis assumes great economic importance since it affects several production aspects. Therefore, knowledge about the occurrence and distribution of this disease is fundamental to adopt the correct prevention measures. The present study aimed to evaluate the frequency of anti-Leptospira spp. antibodies in 24,483 bovine serum samples received between 2007 to 2015 from 21 Brazilian states. Of these, 8,643 (35.3%) were reagents in the microscopic agglutination test to one or more serovars of Leptospira spp. The most frequent serovars were Wolffi (61.47%), Tarassovi (9.62%) and Pomona (7.20%). Hardjo serovar presented a prevalence of 6.27%. Among the 21 states analyzed, the State of Pernambuco had the highest frequency with 88.24% and the State of São Paulo was the origin of the largest number of analyzed samples (13,838), with a frequency of 31.54% of reagents. The results demonstrate a high exposure to several serovars of Leptospira spp. in bovine species in Brazilian states, showing the importance of adopting prophylactic measures in order to reduce the risk of infection in this specie.(AU)

Com o objetivo de avaliar a frequência de anticorpos anti-Leptospira spp., foram analisadas 24.483 amostras de soro sanguíneo bovino, provenientes de 21 estados brasileiros, recebidas no período de 2007 a 2015. Destas, 8.643 (35,3%) foram reagentes no teste de soroaglutinação microscópica a uma ou mais sorovariedades de Leptospira spp., e as sorovariedades com maior frequência foram Wolffi (61,47%), Tarassovi (9,62%) e Pomona (7,20%). A sorovariedade Hardjo apresentou prevalência de 6,27%. Entre os 21 estados analisados, o estado de Pernambuco apresentou a maior frequência, com 88,24%, e o estado de São Paulo foi a origem do maior número de amostras para análise, 13.838, com frequência de 31,54% de reagentes.(AU)

Characterization of adverse reactions to benznidazole in patients with Chagas disease in the Federal District, Brazil

Abstract INTRODUCTION: Benznidazole is used for treating Chagas disease (CD). This cross-sectional study aimed to characterize the adverse drug reactions (ADRs) of benznidazole at a public hospital in Brazil's Federal District. METHODS: Medical records were analyzed and ADRs were categorized by type, intensity, seriousness, and causality. RESULTS: Of the 62 patients who started benznidazole treatment for CD, 41 (66%) presented with 105 ADRs; 23 (37%) discontinued the treatment. Most reactions were classified as probable (81%), severe (63%), serious (67%), and dose-dependent (56%). CONCLUSIONS: The high incidence of ADRs because of treatment withdrawal revealed the need for safer alternatives for CD treatment.

Avaliação das reações de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) na detecção de anticorpos IgG anti-T. gondii em exsudatos cárneos bovinos / Evaluation of indirect Hemagglutination (HI) and Modified Agglutination (MAT) reactions in the detection of anti-T. gondii IgG antibodies in bovine meat exudates

Toxoplasmose é uma zoonose parasitária com ampla distribuição mundial provocada pelo Toxoplasma gondii, considerado um dos protozoários mais bem sucedidos do planeta, pois infecta cerca de um terço da população mundial. Dentre as formas de transmissão, o consumo de carne mal cozida, contendo cistos, tem sido considerado um fator de risco para aquisição desta zoonose. Uma abordagem alternativa para o controle da toxoplasmose pela ingestão de carne bovina seria a sorologia dos bovinos, já que animais soropositivos albergam cistos teciduais. Contudo, a obtenção de soro para esta avaliação, nem sempre é factível, dada a dificuldade de coleta de sangue durante a linha de abate e sua ausência em cortes comerciais. O exsudato cárneo é uma alternativa para detecção de anticorpos anti - T. gondii em cortes comerciais de carne, que foi a proposta deste estudo para avaliar o desempenho dos testes de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) quando comparados ao ELISA usando exsudato cárneo. Este estudo mostrou que a acurácia dos testes de aglutinação não foi viável devido aos baixos índices de sensibilidade e especificidade quando comparados ao ELISA. Estes dados demonstram a importância da escolha de testes eficientes como ELISA para aplicação no controle da qualidade e inocuidade de cortes comerciais de carne bovina.

Toxoplasmosis is a parasitic zoonosis with a wide worldwide distribution caused by Toxoplasma gondii, which is considered one of the most successful protozoa on the planet, since it can infect a third of the world population. Among the forms of transmission, consumption of undercooked meat has been considered as a risk factor for the acquisition of this zoonosis. An alternative approach to toxoplasmosis control by beef ingestion could be the serological diagnosis in cattle, since seropositives animals harbor tissue cysts. However, the use of serum for this evaluation is not always feasible due to the difficulty of blood collection during slaughter and its absence in commercial beef cuts. Meat exudate is an alternative for the detection of anti-T. gondii antibodies in commercial beef cuts, which was the propose of this study to evaluate the performance of Indirect Hemagglutination (HI) and Agglutination Modified (MAT) tests compared to ELISA using meat exudates. This study showed that the agglutination tests accuracy was not viable due to low sensitivity and specificity indexes when compared to ELISA. These data demonstrate the importance of choosing accurate tests such as ELISA for application in quality control and safety of commercial beef cuts.

Sistema de grupos sanguíneos AB em felídeos neotropicais e compatibilidade com gatos domésticos / AB blood group system in neotropical felids and compatibility with domestic cats

O principal sistema de grupos sanguíneos reconhecido para gatos é o AB. Os felinos apresentam anticorpos naturais contra o antígeno do tipo sanguíneo a que não pertencem, o que torna os testes de compatibilidade e as tipagens sanguíneas importantes na prevenção de reações transfusionais. O objetivo deste estudo foi realizar a tipagem sanguínea de oito gatos-mouriscos (Puma yagouaroundi), oito jaguatiricas (Leopardus pardalis), sete gatos-palheiros (Leopardus colocolo), sete gatos domésticos (Felis catus) da raça Persa e oito gatos domésticos sem raça definida (SRD), bem como realizar testes de compatibilidade entre os tipos sanguíneos iguais das diferentes espécies, para avaliar a possibilidade de transfusões interespecíficas. A técnica empregada para a tipagem foi a hemaglutinação em tubos de ensaio. A ocorrência do tipo sanguíneo tipo A foi de 100% entre as jaguatiricas, os gatos-palheiros e os gatos Persas e de 85,72% entre os gatos SRD. A ocorrência do tipo B foi de 100% nos gatos-mouriscos e de 14,28% nos gatos SRD. Considerando os testes de compatibilidade sanguínea, 87,5% (n=4) das jaguatiricas foram incompatíveis com os gatos domésticos, 100% (n= 6) dos gatos-palheiros foram compatíveis com os gatos domésticos e 100% (n= 4) dos gatos-mouriscos foram incompatíveis com os gatos domésticos do tipo B.(AU)

The blood group system recognized for cats is AB. Antibodies against other blood types occur naturally in cats, which makes the compatibility tests and blood typing important for preventing transfusion reactions. Wild felids need blood transfusions in cases of diseases and when run over on highways. The aim of this study was to perform blood typing of eight jaguarundies (Puma yagouaroundi), eight ocelots (Leopardus pardalis), seven pampas cats (Leopardus colocolo), seven domestic cats (Felis catus) of Persian breed and eight non-pedigree domestic cats (Felis catus), and test compatibility among the different species with the same blood types, to evaluate the possibility of performing interspecific blood transfusions. We conducted the study from August to December. We used haemagglutination in test tubes for typing. The occurrence of blood type A was 100% among ocelots, pampas cats and domestic cats of Persian breed, while non-pedigree domestic cats showed 85.72%. The occurrence of type B was 100% for jaguarundis and 14.28% for non-pedigree domestic cats. Regarding blood compatibility tests, 87.5% (n= 4) of the ocelots were incompatible with domestic cats; 100% (n=6) of the pampas cats were compatible with domestic cats, while 100% (n=4) of the jaguarundis were incompatible with type B domestic cats.(AU)

Occurrence of Toxoplasma gondii and risk factors for infection in pigs raised and slaughtered in the Triângulo Mineiro region, Minas Gerais, Brazil / Ocorrência e fatores de risco da infecção por Toxoplasma gondii em suínos criados e abatidos na região do Triângulo Mineiro, Minas Gerais, Brasil

The Triângulo Mineiro region from Minas Gerais state, is an important meat-exporting region of Brazil and data about Toxoplasma gondii infection in pigs raised and slaughtered in this area are scarce. Therefore, the aim of this study was to evaluate the occurrence of T. gondii in swine and establish the risk factors associated with the infection. Samples were collected from 600 pigs raised under intensive system in farms located at three different counties (Carmo do Paranaíba, Patrocínio and Perdizes). The samples were submitted to indirect hemagglutination antibody test with dilution of 1:32 and to indirect immunofluorescence antibody test with a cutoff of 1:64. The occurrence of positive pig was 3.3% (n=20) and 51.8% (n=311) respectively. A significant difference was observed between toxoplasmatic infection and factors such as lineage, animal origin, size of the farm, collective raising with others species, presence of rodents and type of water offered (p≤0.05). There was no difference between gender and the farm goals. The results demonstrated an occurrence of anti-T.gondii antibodies higher than expected for intensive pig raising system on the studied area, which could indicate a possible sanitary management problem on the studied proprieties. Improvements on the raising techniques are necessary to reduce T. gondii infection sources.(AU)

A região do Triângulo Mineiro, no estado de Minas Gerais, é uma importante região exportadora de carne do Brasil e pesquisas sobre a infecção por Toxoplasma gondii em suínos criados e abatidos nesta região são escassos. Portanto, o objetivo deste estudo foi avaliar a ocorrência de T. gondii nesses animais e estabelecer os fatores de risco associados com a infecção. Foram coletadas amostras de 600 suínos criados sob sistema intensivo, em fazendas localizadas em três municípios diferentes (Carmo do Paranaíba, Patrocínio e Perdizes). As amostras foram submetidas à Hemaglutinação Indireta com diluição de 1:32 e à Reação de Imunofluorescência Indireta com ponto de corte 1:64. A ocorrência de suínos positivos foi de 3,3% (n=20) e 51,8% (n=311), respectivamente. Foi observada diferença significativa entre a infecção toxoplásmica e fatores como linhagem, procedência dos animais, tamanho das propriedades, criação em conjunto com outras espécies, presença de roedores e tipo de água consumida (p≤0,05). Não houve diferenças estatísticas entre o sexo e finalidade de produção em relação à infecção por T. gondii. Os resultados demonstraram uma ocorrência de anticorpos anti-T. gondii superior à esperada em criações intensivas de suínos na região estudada, o que poderia indicar uma possível falha no manejo sanitário das propriedades estudadas. Melhorias nas técnicas de criação são necessárias para redução das fontes de infecção por T. gondii nos rebanhos.(AU)

Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Detection, purification and characterization of a lectin from freshwater green algae Spirogyra spp

ABSTRACT Freshwater algae are rich sources of structurally biologically active metabolites, such as fatty acids, steroids, carotenoids and polysaccharides. Among these metabolites, lectins stand out. Lectins are proteins or glycoproteins of non-immune origin which bind to carbohydrates or glycoconjugates, without changing ligand structure. Many studies have reported on the use of Spirogyra spp. as effective bioindicators of heavy metals; however, reports on Spirogyra molecular bioprospecting are quite limited. Therefore, this study aimed to detect, isolate, purify and characterize a lectin present in the freshwater green algae Spirogyra. Presence of the lectin protein in the extract was detected by hemagglutination assays. Subsequently, the protein extract was subjected to a sugar inhibition assay to identify the lectin-specific carbohydrate. Following this, the extract was applied to a guar gum column to afford the pure lectin. The lectin was inhibited by N-acetyl-glucosamine and N-acetyl-beta-D-mannose, but more strongly by D-galactose. The apparent molecular mass of the purified lectin was evaluated by Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Electrophoretic analysis revealed a single protein band with an apparent molecular mass of 56 kDa. Thus, it could be concluded that a lectin was purified from Spirogyra spp.

Seroprevalence of Toxoplasma gondii Infection in Wild Boars, Wild Rabbits, and Wild Chickens in Hubei Province, China

Toxoplasma gondii causes serious infection worldwide in humans and animals. In this study, the seroepidemiology of toxoplasmosis was investigated in wild boars (Sus scrofa) (n=377), wild rabbits (cape hare, Lapus capensis) (n=331), and wild chickens (red junglefwol, Gallus gallus) (n=571) in 4 forested and country sided area of Hubei province of China. For this, blood samples were collected and tested by indirect hemagglutination test (IHA). The seroprevalence was found to be 7.2%, 5.1%, and 12.6% in wild boars, rabbits, and chickens, respectively, with significant differences among these species. The prevalence of T. gondii infection in male and female wild boars was found to be 7.9% and 6.5% (P<0.01), in male and female rabbits was 5.6% and 4.9% (P<0.01), and in male and female chickens was 17.1% and 7.7% (P<0.01), respectively, with significant differences between 2 genders of chickens (P<0.01). The findings of this study may help in planning of the prevention measures against T. gondii infection in wild animals in this area.

Isolation and Genotyping of Toxoplasma gondii in Brazilian Dogs

Strains of Toxoplasma gondii in Brazil are highly genetically diverse compared to strains from North America and Europe. Dogs are epidemiologically important because they act as sentinels for T. gondii infections in humans and are good indicators of environmental contamination. The aim of this study was to isolate and genetically characterize T. gondii strains from tissues of naturally infected Brazilian dogs. For this study, 21 blood samples were collected from dogs at the Zoonosis Control Centers of Ilhéus and Itabuna cities, Bahia, Brazil. The sera were examined for T. gondii antibodies using the indirect hemagglutination test. Brains and hearts of seropositive dogs were bioassayed in mice to isolate and characterize T. gondii parasites by PCR-RFLP using 10 genetic markers (SAG1, newSAG2, SAG3, BTUB, c22-8, c29-2, GRA6, PK1, APICO, and L358). However, T. gondii was isolated from only 4 (57.1%) dogs, designated TgDgBr6, 13, 17, and 21. All strains were virulent, causing clinical changes (rough hair coat, lethargy, and abdominal distention) and the death of all mice within 8–20 days after inoculation. Genetic analysis of these 4 T. gondii isolates revealed 4 distinct genotypes with different clonal lineage combinations (types I, II, and III) and 2 atypical alleles. Using PCR-RFLP with several markers, this study contributes to evaluations of the genetic diversity of strains circulating in Brazil.

Viral type characterization and clinical aspects of canine parvovirus in naturally infected dogs in São Paulo State, Brazil / Caracterização viral e aspectos clínicos de parvovirose em cães naturalmente infectados no Estado de São Paulo

Since the first isolation of canine parvovirus type 2 (CPV-2) in late 70's new virus types as CPV-2a and CPV-2b have been emerged and becoming prevalent in natural canine population and more recently, a third subtype was identified , CPV-2c. The main purpose of this study was to detect and characterize canine parvovirus currently present in Central-West region of São Paulo state, in Brazil. Fecal samples were collected of vaccinated and non-vaccinated dogs, clinically suspected of having CPV infection brought to the Infectious Diseases Service, Veterinary Hospital of FMVZ-UNESP. All samples (n=30) were screening for canine parvovirus through hemagglutination test and those resulting as positive (n=20) were submitted to PCR and the products were subsequently sequenced for subtype characterization. Results were tested for association with age, hematological values, viral hemagglutination titers in the feces, vaccination status and survival. Leukopenia was found in all animals, death occurred in 30% of unvaccinated dogs and in 42% of vaccinated ones. In a total of 20 positive sequenced samples, 18 were classified as CPV-2b, one as CPV-2c, and one as CPV-2a, being CPV2a and CPV2c detected in unvaccinated puppies. Compared to the reference samples amino acid change at position 426 in those circling virus was identified. The study results demonstrate the predominance of CPV-2b and the presence of CPV-2a and CPV-2c in naturally infected, vaccinated and unvaccinated dogs in in São Paulo region.(AU)

Desde o primeiro isolamento do parvovirus canino tipo 2 (CPV-2) no final dos anos 70 novos subtipos virais como CPV-2a e CPV-2b surgiram e foram se tornando prevalentes na população canina; posteriormente um terceiro subtipo foi identificado, CPV- 2-C. O principal objetivo deste estudo foi detectar e caracterizar os subtipos de parvovírus canino atualmente presente na região Centro-Oeste do Estado de São Paulo-Brasil. Amostras de fezes foram coletadas de cães vacinados e não vacinados, atendidos no Serviço de Enfermidades Infecciosas dos Animais, Hospital Veterinário da FMVZ-UNESP, com suspeita clínica parvovirose . Todas as amostras (n = 30) foram submetidas teste de hemaglutinação para parvovirus canino e as positivas (n = 20) submetidas a PCR; os produtos amplificados foram subsequentemente sequenciados para caracterização do subtipo viral. Os resultados foram associados com a idade, os valores hematológicos, os títulos de hemaglutinação viral nas fezes, estado de vacinação e sobrevivência. A leucopenia foi encontrada em todos os animais; Obito foi observado em 30% dos cães não vacinados e 42% dos vacinados. Em um total de 20 amostras positivas sequenciadas, 18 foram classificadas como CPV-2b, uma como CPV-2c, e uma como CPV-2a. CPV 2a e CPV2c foram detectados em filhotes não vacinados. Em comparação com a amostra de referência foi evidenciada uma mudança de aminoácido na posição 426 nas amostras virais circulantes. Os resultados do estudo demonstram a predominância de CPV-2b e a presença de CPV-2a e CPV-2c em cães naturalmente infectados, vacinados e não vacinados na região de São Paulo.(AU)

Lectin activity in mycelial extracts of Fusarium species

ABSTRACT Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to D-ribose, L-fucose, D-glucose, L-arabinose, D-mannitol, D-galactosamine hydrochloride, D-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-D-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age.

Frecuencia de antígenos del sistema sanguíneo Rh y del sistema Kell en donantes de sangre / Frequency of antigens in Rh and Kell blood system in blood donors

Introducción: los estudios inmunohematológicos que se realizan a los donantes de sangre se orientan a proporcionar al receptor una terapia transfusional compatible con el sistema sanguíneo ABO y antígeno D del sistema Rh. Sin embargo, como una forma de incrementar la seguridad transfusional, surge el interés de ampliar la gama de antígenos a determinar y por ende, a compatibilizar previo a una transfusión sanguínea. Objetivo: determinar la frecuencia de los cinco antígenos mayores del sistema Rh y los antígenos K1 y K2 del sistema Kell en donantes voluntarios de sangre. Métodos: Estudio descriptivo transversal que incluyó 200 donantes voluntarios de sangre del Centro Productivo Regional de Sangre del Maule (CPRSM) seleccionados al azar. Se realizó fenotipificación de los cinco antígenos mayores del sistema Rh. y el antígeno K1 y K2 del sistema Kell. Se utilizó la técnica de hemaglutinación en tubo, con sueros monoespecíficos y DG Gel® Coombs. Se calculó la frecuencia fenotípica de los antígenos D, C, c, E y e del sistema Rh., y K1 y K2 del sistema Kell, en porcentajes. A partir de la frecuencia de los fenotipos Rh. se determinó la frecuencia del genotipo más probable de dicho sistema. Para el Kell se estimó el genotipo en base al fenotipo. Resultados: sistema Rh: 96 por ciento de las muestras estudiadas presentaba el antígeno D, 97,5 por ciento el antígeno e; 35,5 por ciento el antígeno E; 79 por ciento el antígeno C y 65,5 por ciento el antígeno c. El genotipo más frecuente fue CDe/CDe. Sistema Kell: se encontró una frecuencia del 4 por ciento para el antígeno K1, mientras que el antígeno K2 presenta una frecuencia del 99,5 por ciento. Al nivel de frecuencia genotípica se detectó que el 96 por ciento de la población tiene un genotipo homocigoto para K2 (kk). Conclusiones: La frecuencias de los siete antígenos estudiados es similar a la descrita en otras poblaciones(AU)

Introduction: Immunologic studies performed on blood donors are directed to provide a transfusion therapy compatible with ABO blood group system and Rh system D antigen in the recipient. However, as a way to increase transfusion safety, the interest of expanding the range of antigens to determine and therefore to be tested for compatibility prior to a blood transfusion, arises. Aim: To determine the frequency of the five major antigens of Rh. system and K1 and K2 antigens of the Kell system in blood donors. Methods: Cross-sectional study including 200 randomly selected voluntary blood donors from Centro Productivo Regional de Sangre del Maule (CPRSM). Phenotyping of five major antigens of Rh. system and K1 and K2 Kell system antigens was carried out. The tube hemagglutination technique with monospecific Coombs sera and DG Gel ® was used. The Rh. system D, C, c, E, e antigens and Kell system K1 and K2 antigen phenotypic frequencies were calculated in percentages. The most likely Rh.genotype was determined from the phenotype frequency of this system. Similarly, in Kell system the genotype frequency was determined based on phenotype. Results: In the Rh.system, 96 percent of the samples studied had D antigen; 97.5 percent had the e antigen, and 35.5 percent the E antigen. Antigen C was present in 79 percent and c in 65.5 percent. The most frequent Rh. genotype was CDe/CDe. In Kell system, K1 antigen presented a frequency of 4 percent, while antigen K2 presented a 99.5 percent. Regarding genotypic frequency, a 96 percent of the population showed a K2 (kk) homozygous genotype(AU) Conclusion: The frequency of the seven antigens studied is similar to that described in other populations(AU)

Prevalence and Genetic Characterization of Toxoplasma gondii in Pet Dogs in Central China

The prevalence and genotype of Toxoplasma gondii infection in dogs in Henan Province, Central China was investigated. A total of 125 blood samples were collected from pet dogs during April to June 2013, and all samples were examined by indirect hemagglutination antibody test (IHA) and nested PCR. The overall T. gondii prevalence in pet dogs was 24.0% (30/125), with 20.8% (26/125) in IHA and 10.4% (13/125) in PCR, respectively. No statistical associations were found between animal gender and age and the prevalence of T. gondii infection. Thirteen positive DNA samples were genotyped using 11 PCR-RFLP markers, including SAG1, (3'+5') SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Of these, only 2 samples were genotyped with complete data for all loci, and a novel genotype (type III at SAG3 and GRA6 loci, and type I at other loci) was identified. This is the first report of genetic characterization of T. gondii infection in dogs in China.

Prevalence and Genetic Characterization of Toxoplasma gondii in Pet Dogs in Central China

The prevalence and genotype of Toxoplasma gondii infection in dogs in Henan Province, Central China was investigated. A total of 125 blood samples were collected from pet dogs during April to June 2013, and all samples were examined by indirect hemagglutination antibody test (IHA) and nested PCR. The overall T. gondii prevalence in pet dogs was 24.0% (30/125), with 20.8% (26/125) in IHA and 10.4% (13/125) in PCR, respectively. No statistical associations were found between animal gender and age and the prevalence of T. gondii infection. Thirteen positive DNA samples were genotyped using 11 PCR-RFLP markers, including SAG1, (3'+5') SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Of these, only 2 samples were genotyped with complete data for all loci, and a novel genotype (type III at SAG3 and GRA6 loci, and type I at other loci) was identified. This is the first report of genetic characterization of T. gondii infection in dogs in China.

Diagnostic reliability of an immunochromatographic test for Chagas disease screening at a primary health care centre in a rural endemic area

Many patients with Chagas disease live in remote communities that lack both equipment and trained personnel to perform a diagnosis by conventional serology (CS). Thus, reliable tests suitable for use under difficult conditions are required. In this study, we evaluated the ability of personnel with and without laboratory skills to perform immunochromatographic (IC) tests to detect Chagas disease at a primary health care centre (PHCC). We examined whole blood samples from 241 patients and serum samples from 238 patients. Then, we calculated the percentage of overall agreement (POA) between the two groups of operators for the sensitivity (S), specificity (Sp) and positive (PPV) and negative (NPV) predictive values of IC tests compared to CS tests. We also evaluated the level of agreement between ELISAs and indirect haemagglutination (IHA) tests. The readings of the IC test results showed 100% agreement (POA = 1). The IC test on whole blood showed the following values: S = 87.3%; Sp = 98.8%; PPV = 96.9% and NPV = 95.9%. Additionally, the IC test on serum displayed the following results: S = 95.7%; Sp = 100%; PPV = 100% and NPV = 98.2%. Using whole blood, the agreement with ELISA was 96.3% and the agreement with IHA was 94.1%. Using serum, the agreement with ELISA was 97.8% and the agreement with IHA was 96.6%. The IC test performance with serum samples was excellent and demonstrated its usefulness in a PHCC with minimal equipment. If the IC test S value and NPV with whole blood are improved, then this test could also be used in areas lacking laboratories or specialised personnel.

Estandarización de la técnica de aglutinación directa para el inmunodiagnóstico de la enfermedad de Chagas / Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease

Introducción. La enfermedad de Chagas es causada por el parásito Trypanosoma cruzi y su diagnóstico inmunológico se basa principalmente en la detección de anticuerpos contra T. cruzi mediante pruebas tales como ELISA, inmunofluorescencia indirecta (IFI) y hemaglutinación indirecta (HAI). Esta última tiene el inconveniente de requerir la preparación de eritrocitos de carnero, difíciles de obtener y de poca duración. Sin embargo, existen pruebas alternativas, como la técnica de aglutinación directa. Objetivo. Estandarizar la técnica de aglutinación directa para el diagnóstico de la enfermedad de Chagas. Materiales y métodos. Se prepararon parásitos epimastigotes de T. cruzi mediante dos protocolos, con tratamiento con tripsina y sin él. Los parásitos se colorearon, y se determinaron las condiciones óptimas de concentración parasitaria y diluciones de suero. Se utilizaron sueros de pacientes con enfermedad de Chagas, de individuos sanos y con otras parasitosis. Resultados. La concentración parasitaria óptima fue de 500 x10 6 parásitos/ml, utilizando parásitos coloreados y sin tratamiento con tripsina. Las diluciones de suero óptimas fueron de 1/25, 1/50 y1/100, y el punto de corte, la dilución de 1/50. La técnica estandarizada mostró índices diagnósticos de sensibilidad de 94,3 % (IC 95% 79,5-99,0) y de especificidad de 96,3 % (IC 95% 88,8-99,0); se encontró reacción cruzada en tres sueros de individuos con leishmaniasis visceral, con valores pronósticos positivo y negativo de 91,7 % (IC 95% 76,4-97,8) y de 97,5 % (IC 95% 90,4-99,6), respectivamente. Se compararon los resultados con los obtenidos por HAI, ELISA e IFI y la concordancia fue de 96 % con un índice kappa de 0,90 (IC 95% 0,81-0,99). Conclusión. La técnica de aglutinación directa estandarizada podría ser útil para el inmunodiagnóstico de la enfermedad de Chagas.

Introduction: Chagas´ disease is caused by the parasite Trypanosoma cruzi and its immunological diagnosis is mainly based on the detection of antibodies against T. cruzi using tests such as the ELISA, the indirect fluorescence antibody test (IFAT) and the indirect hemagglutination test (IHAT). The main disadvantage of the IHAT is the need to prepare sheep erythrocytes, whose availability is limited and they have a short duration once prepared. However, there are alternative tests, such as the direct agglutination test (DAT). Objective: To standardize the direct agglutination test for the diagnosis of Chagas disease. Materials and methods: Trypanosoma cruzi epimastigotes were prepared using two protocols, with and without trypsin treatment. The parasites were stained and optimal conditions for parasitic concentration and serum dilutions were determined. We evaluated the technique using sera from patients with Chagas disease, from healthy individuals and from individuals with other parasitic diseases. Results: The optimal parasitic concentration was 500 x 10 6 parasites/ml using stained parasites without trypsin treatment. The optimal serum dilutions were 1/25, 1/50 y 1/100 and the cut-off point was the 1/50 dilution. The diagnostic indices for the standardized technique were as follows: Sensitivity, 94.3% (95% CI: 79.5-99.0) and specificity, 96.3% (95% CI: 88.8-99.0), with positive and negative predictive values ?? of 91.7% (95% CI: 76.4-97.8) and 97.5% (95% CI: 90.4-99.6), respectively. Cross-reaction was observed only in three sera from individuals with visceral leishmaniasis. The results were compared with those obtained by IHA, ELISA, and IFA, and the concordance rate was 96% and the kappa index, 0.90 (95% CI: 0.81-0.99). Conclusion: The standardized direct agglutination test could be useful for immunodiagnosis of Chagas disease.

SEROPREVALENCE OF T. cruzi INFECTION IN BLOOD DONORS AND CHAGAS CARDIOMYOPATHY IN PATIENTS FROM THE COAL MINING REGION OF COAHUILA, MEXICO / Soroprevalência da infecção pelo T. cruzi em doadores de sangue e cardiomiopatia chagásica em pacientes da região carbonífera de Coahuila, México

Context and Objective: Chagas disease is considered a worldwide emerging disease; it is endemic in Mexico and the state of Coahuila and is considered of little relevance. The objective of this study was to determine the seroprevalence of T. cruzi infection in blood donors and Chagas cardiomyopathy in patients from the coal mining region of Coahuila, Mexico. Design and Setting: Epidemiological, exploratory and prospective study in a general hospital during the period January to June 2011. Methods: We performed laboratory tests ELISA and indirect hemagglutination in three groups of individuals: 1) asymptomatic voluntary blood donors, 2) patients hospitalized in the cardiology department and 3) patients with dilated cardiomyopathy. Results: There were three levels of seroprevalence: 0.31% in asymptomatic individuals, 1.25% in cardiac patients and in patients with dilated cardiomyopathy in 21.14%. Conclusions: In spite of having detected autochthonous cases of Chagas disease, its importance to local public health remains to be established as well as the details of the dynamics of transmission so that the study is still in progress.

Contexto e Objetivo: A doença de Chagas é mundialmente considerada uma doença emergente, é endêmica no México e no estado de Coahuila e considerada de pouca relevância. O objetivo do estudo foi determinar a soroprevalência da infecção pelo T. cruzi em doadores de sangue e cardiomiopatia chagásica em pacientes da região carbonífera de Coahuila, México. Desenho e Local: Estudo epidemiológico, exploratório e prospectivo em um hospital geral no período de janeiro a junho de 2011. Métodos: Foram realizados testes de laboratório ELISA e hemoglutinação indireta em três grupos de indivíduos: 1) doadores de sangue voluntários assintomáticos, 2) pacientes internados na área de cardiologia e 3) pacientes com cardiomiopatia dilatada. Resultados: Foram achados três níveis de soroprevalência: 0,31% em indivíduos doadores de sangue assintomáticos, 1,25% em pacientes cardiopatas e, em pacientes com cardiomiopatia dilatada 21,14%. Conclusão: Detectamos casos autóctones de doença de Chagas em área considerada não endêmica. Deve ser determinada sua importância na saúde pública regional e local, para estabelecer os detalhes do mecanismo de transmissão. O estudo ainda está em desenvolvimento.

Comparison of seven diagnostic tests to detect Trypanosoma cruzi infection in patients in chronic phase of Chagas disease / Comparación de siete pruebas diagnósticas para detectar infección por Trypanosoma cruzi en pacientes en fase crónica de la enfermedad de Chagas

Objective: To compare the diagnostic performance of seven methods to determine Trypanosoma cruzi infection in patients with chronic Chagas disease. Methods: Analytical study, using the case-control design, which included 205 people (patients with Chagasic cardiomyopathy, n= 100; control group, n= 105). Three enzyme linked immunosorbent assays, one indirect hemagglutination assay and one immunochromatographic test were assessed. Additionally, DNA amplification was performed via the PCR method using kinetoplast and nuclear DNA as target sequences. For the comparative analysis of diagnostic tests, the parameters used were sensitivity, specificity, positive and negative predictive values, Receiver Operator Characteristic (ROC), positive and negative likelihood ratio, as well as κ quality analysis. Results: The commercial Bioelisa Chagas test showed the highest sensitivity (98%), specificity (100%), and positive and negative predictive values; additionally it had the highest discriminatory power. Otherwise, the amplification of T. cruzi DNA in blood samples showed low values of sensitivity (kinetoplast DNA= 51%, nuclear DNA= 22%), but high values of specificity (100%), and moderate to low discriminatory ability. Conclusion: The comparative analysis among the different methods suggests that the diagnostic strategy of T. cruzi infection in patients with chronic Chagas disease can be performed using ELISA assays based on recombinant proteins and/or synthetic peptides, which show higher diagnosis performance and can confirm and exclude the diagnosis of T. cruzi infection. The molecular methods show poor performance when used in the diagnosis of patients with chronic Chagas disease.

Objetivo: Comparar la capacidad diagnóstica de siete métodos para determinar infección por Trypanosoma cruzi, en pacientes con enfermedad de Chagas crónica. Métodos: Estudio analítico de casos y controles, que incluyó 205 personas (pacientes con miocardiopatía chagásica, n= 100; grupo control, n= 105). Se evaluaron tres inmunoensayos enzimáticos, una hemaglutinación indirecta y una inmunocromatografia. Adicionalmente, se realizó amplificación de ADN de T. cruzi por reacción en cadena de la polimerasa utilizando como secuencias diana ADN de kinetoplasto y nuclear. Para el análisis comparativo de las pruebas diagnósticas, los parámetros utilizados fueron sensibilidad, especificidad, valores predictivo positivo y negativo, análisis ROC, razón de verosimilitud positiva y negativa, así como análisis de calidad κ. Resultados: La prueba Bioelisa para Chagas mostró la mayor sensibilidad (98%), especificidad (100%) y valores predictivos positivo y negativo; además ésta tuvo el mayor poder discriminatorio. En contraste, los ensayos de amplificación de ADN de T. cruzi mostraron baja sensibilidad (ADN de kinetoplasto= 51%, ADN nuclear= 22%), alta especificidad (100%) y de moderada a baja capacidad discriminatoria. Conclusión: El análisis comparativo entre los métodos sugiere utilizar como estrategia diagnóstica en pacientes crónicos con enfermedad de Chagas, los ensayos de ELISA con proteínas recombinantes y/o péptidos sintéticos por mostrar un rendimiento diagnóstico superior y tener la capacidad de confirmar y descartar el diagnóstico de infección por T. cruzi. Los métodos moleculares muestran pobre rendimiento para ser utilizados en el diagnóstico de pacientes en fase crónica con enfermedad de Chagas.

Evaluation of the Chagas Disease Control Program in Açucena Municipality, Rio Doce Valley, State of Minas Gerais, Brazil

Introduction Açucena Municipality, Rio Doce Valley, State of Minas Gerais, Brazil temporarily (2001-2005) interrupted epidemiological surveillance for Chagas disease. The objective of this work was to evaluate the Chagas Disease Control Program (CDCP) in Açucena and to offer suggestions for improving local epidemiological surveillance. Methods This study was conducted in three phases: I) a serological investigation of schoolchildren aged 5 to 15 years using an enzyme-linked immunosorbent assay (ELISA) test performed on blood collected on filter paper followed by ELISA, indirect immunofluorescence (IIF) and indirect hemaglutination (IHA) on venous blood for borderline cases and those in the gray zone of reactivity; II) vector evaluation using the data obtained by local health agents during 2006-2010; and III) examination by ELISA, IIF and IHA of serum samples from the inhabitants of houses where infected Triatoma vitticeps was found and evaluation of their knowledge about Chagas disease. Results Five individuals had inconclusive results in the ELISA screening but were seronegative for Chagas disease. The triatomine evaluation revealed the presence of three species: Triatoma vitticeps, Panstrongylus megistus and Panstrongylus diasi. Triatoma vitticeps was the most prevalent and widespread, with a higher (67%) index of Trypanosoma cruzi flagellates and evidence of colonization. Most of the inhabitants of the infested houses recognized triatomines and had basic knowledge about Chagas disease. Conclusions Although T. vitticeps is not clearly associated with Chagas disease transmission, these results highlight the importance of maintaining CDCP in endemic areas and the need for greater emphasis on epidemiological surveillance, especially in areas with important vectorial changes or that have been modified by human intervention. .