Feasibility of Indirect immunofluorescence (IIF) alone as a screening method for antinuclear antibody in connective diseases in India's sub-Himalayan region

Background: For the management of connective tissue disorders (CTDs), antinuclear antibody (ANA) testing is essential, both from diagnostic and prognostic points of view. Usually, patterns obtained by ANA-IIF testing correlates to specific autoantibodies as obtained from the test for ENA (by LIA/ELISA, etc.). But to apply these data from western studies, we may need validation in the local population like our subjects in sub-Himalayan (Garhwal region) area where CTDs are common. Also, suppose ANA-IFA pattern's correlation is reliably known in our population, it can minimize the cost of managing CTDs by limiting ENA testing, which is 10 times costlier than ANA-IIF. Hence, this study was undertaken to know the specific autoantibody targets (ENA by LIA) against ANA-IIF patterns in our local population. Materials and Methods: In this retrospective cross-sectional work, serum samples of CTDs were tested for ANA by IIF (Euroimmune AG) and ENA by LIA (Euroline ANA-3G) continuously for 36 months. The manufacturer's kit insert was followed, and results were analyzed applying appropriate statistical methods. Results: Major ANA-IIF patterns were found to be associated with specific autoantibodies, for example, Nuclear homogenous with dsDNA, nucleosomes, histones; speckled pattern with nRNP/Sm, Sm, SSA/Ro-52, SSB; nucleolar pattern with Scl-70, Pm-Scl 100 and centromere pattern with CENP-B. Anticytoplasmic (ACA) are found to be linked with some ANA negative (by IIF) samples, emphasizing the need for careful observation for ACA especially where ANA is not found. Conclusions: In most subjects, specific ENA targets correlated well with ANA-IIF patterns, implying effective cost minimization in CTD management. Similar future prospective studies (with clinical data) can provide a database and reference for our population.

Cloning, expression and purification of Plasmodium knowlesi circumsporozoite protein and immunoblot analysis with P. knowlesi strain A1H1 protein extract

@#Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite’s erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.

Analysis of reactive HCV results detected by current blood screening strategy / 中国输血杂志

【Objective】 To assess the status of HCV infection by analyzing the results of anti-HCV reactive blood samples detected by the current blood testing strategy, and discuss the viability of classified management of reactive blood donors. 【Methods】 The anti-HCV reactive samples (dual ELISA and once NAT), from May 2017 to October 2018, were divided into three groups: samples both anti-HCV and HCV RNA reactive, sole HCV RNA reactive, and sole anti-HCV reactive, and all of them were confirmed by recombinant immunoblot assay (RIBA). The positive predictive value (PPV) between groups were compared. The sensitivity, specificity and PPV for each reagent under different screening threshold (screening threshold for routine detection, optimal screening threshold, and corresponding screening threshold of the highest PPV) were analyzed. The group with low PPV were stratified by ELISA S/CO values, and PPV by different screening threshold was compared. 【Results】 There were 939 reactive samples (0.49%, 937/191 627). Confirmed by RIBA, the positive rate of anti-HCV reactive samples was 10.67%(100/937). Two samples were sole HCV RNA reactive (0.001%). Both anti-HCV+ HCV RNA reactive samples were 6.71%(63/939), with the PPV of 96.83%(61/63). Sole anti-HCV reactive samples were 93.08(874/939), with the PPV of 4.46%(39/874), among which PPV by dual and one ELISA reagent were 18.72% and 0.15%, respectively, showing statistically significant difference (P<0.05). The PPV between different S/CO values was statistically significant (P<0.05). The optimal screening thresholds of anti-HCV reagent were 9.29 and 3.97, according to the ROC curve, with significant difference noticed in PPV by different screening threshold (P<0.05). PPV in the sole anti-HCV reactive group increased from 4.46% (the routine screening threshold) to 49.35%(the optimal screening threshold), and the difference was statistically significant (P<0.05). 【Conclusion】 The blood donors with both anti-HCV and HCV RNA reactive can be determined as HCV infection and need to be permanently deferred. The S/CO value of sole anti-HCV reactive samples was positively correlated with RIBA confirmation results, and the higher the S/CO value, the greater the chances of positive confirmation are. With the current blood screening strategy, the HCV infection status of sole anti-HCV reactive blood donors can be determined by establishing a screening threshold with high PPV or adding confirmatory test.

Relación entre los anticuerpos contra antígenos extraíbles del núcleo y las enfermedades del tejido conectivo identificados por Immunoblots en un hospital y universidad de Lima / Relationship between antibodies against extractable nucleus antigens and connective tissue diseases identified by Immunoblot in a hospital in Lima, Peru

Objetivos: Determinar la relación de los anticuerpos con los antígenos del núcleo extraíble y las enfermedades del tejido conectivo identificadas por Immunoblot en un hospital de Lima, Perú. Material y métodos: Estudio de tipo observacional, ciencias básicas, analíticas y transversales, realizado en el Servicio de Inmunología del Hospital Nacional Arzobispo Loayza entre enero de 2018 y junio de 2018. Analizamos 291 historias clínicas de pacientes con enfermedad del tejido conectivo y para la detección de anticuerpos contra los antígenos extraíbles del núcleo se empleó el método de Immunoblots. Resultados: La frecuencia de los anticuerpos contra antígenos nucleares extraíbles en pacientes con enfermedad del tejido conectivo identificados por Immunoblot fue 789 (100%). Se demostró que existe una relación significativa p <0.05 de Anti-histonas (X2 = 64.19; p = 0,000), anti-nucleosomas (X2 = 71,16; p = 0,000), anti-dsDNA (X2 = 71,44; p = 0,000), anti-SM (X2 = 10,08; p = 0,003) y lupus eritematoso sistémico con prueba de Chi-cuadrado de Pearson. Se demostró que existe una relación significativa p <0.05 del Anti-SSA (X2 = 61,33; p = 0.001), anti-SSB (x2 = 51,00; p = 0.001), anti-Ro 52 (X2 = 62,60; p = 0,000) y síndrome de Sjogren con prueba de Chi-cuadrado de Pearson. Se demostró que existe una relación significativa p <0.05 de Anti-CENP B (p = 0.001) y calcinosis, fenómeno de Raynaud, dismotilidad esofágica, esclerodactilia y Telangiectasia (CREST) con Fisher. Conclusiones: Existe relación de anticuerpos con antígenos de núcleo extraíbles y lupus eritematoso sistémico, síndrome de Sjogren, enfermedad mixta del tejido conectivo, enfermedad del CREST, esclerodermia y polimiositis.

Objectives: To determine the relationship of antibodies to extractable nucleus antigens and connective tissue diseases identified by Immunoblot in a hospital in Lima, Peru. Material and methods: Study of the observational type, basic sciences, analytical and transversal, carried out in the Immunology service of the national Hospital Archbishop Loayza between January 2018 and June 2018. We analyzed 291 clinical histories of patients with connective tissue disease and for the detection of antibodies to the extractable antigens of the nucleus the method of Immunoblot was employed. Results: The frequency of the antibodies against extractable nuclear antigens in patients with connective tissue disease identified by Immunoblot was 789 (100%). It was demonstrated that there is significant relationship p < 0.05 of Anti-histones (X2 = 64.19; p = 0,000), anti-nucleosomas (X2 = 71,16; p = 0,000), anti-dsDNA (X2 = 71,44; p = 0,000), anti-SM (X2 = 10,08; p = 0,003) and Lupus Systemic erythematosus with Pearson Chi-square test. It was demonstrated that there is significant relationship p < 0.05 of the Anti-SSA (X2 = 61,33; p = 0.001), anti-SSB (X2 = 51,00; p = 0.001), anti-Ro 52 (X2 = 62,60; p = 0,000) and Sjogren's syndrome with Pearson Chi-square test. It was demonstrated that there is significant relationship p < 0.05 of Anti-CENP B (p = 0.001) and calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and Telangiectasia (CREST) with exact Fisher statistician. Conclusions: There is a relationship of antibodies to extractable nucleus antigens and systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease, calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and Telangiectasias (CREST), Scleroderma and Polymyositis.

Low prevalence of infection by Sarcocystis neurona in horses from the State of Alagoas, Brazil / Baixa prevalência da infecção por Sarcocystis neurona em cavalos do estado de Alagoas, Brasil

Abstract The aim of this study was to determine the prevalence of infection by Sarcocystis neurona in horses and identify potential risk factors. Were analyzed 427 samples from 36 farms in 21 municipalities in the Alagoas State, Brazil. Presence of anti-S. neurona antibodies was diagnosed by indirect immunofluorescence antibody test (IFAT) and was confirmed using the immunoblot test. Risk factors were assessed through investigative questionnaires on animal management on the farms. The prevalence of anti-S.neurona antibodies was 2.8% (confidence interval, CI: 1.5-4.9%) from IFAT and 1.6% (CI:0.8-3.34%) from immunoblot, and there were positive horses on 16.6% of the studied farms. None of the variables studied presented associations with serological status for S. neurona. This is the first report on infection by S. neurona in horses reared in Alagoas, Brazil showing a low exposure to S. neurona in this region, but with significant numbers of foci.

Resumo Objetivou-se neste estudo determinar a prevalência e os fatores de risco associados à infecção por Sarcocystis neurona em equinos. Foram analisadas 427 amostras de 36 propriedades localizadas em 21 municípios do estado de Alagoas. O diagnóstico de anticorpos anti-S. neurona foi realizado pela técnica de Imunofluorescência Indireta (IFI) e confirmada por immunoblot. O estudo dos fatores de risco foi realizado a partir de questionários investigativos sobre o manejo dos animais nas propriedades. A prevalência de anticorpos anti-S. neurona foi de 2,8% (I.C. 1,5-4,9%) na IFI e de 1,6% (I.C. 0,8-3,34%) no immunoblot com equinos positivos em 16,6% das propriedades estudadas. Nenhuma variável estudada apresentou associação com o status sorológico para S. neurona. Este é o primeiro relato da infecção por S. neurona em equinos criados no Estado de Alagoas, Brasil, confirmando que os animais desta região têm baixa exposição a S. neurona, mas com significativo número de focos.

The Role of the Signal-to-Cutoff Ratio in Automated Anti-HCV Chemiluminescent Immunoassays by Referring to the Nucleic Acid Amplification Test and the Recombinant Immunoblot Assay

BACKGROUND: Following discontinuation of the recombinant immunoblot assay (RIBA), the only available supplementary test for the detection of hepatitis C virus (HCV) is the nucleic acid amplification test (NAAT). However, the NAAT does not adequately detect past HCV. Consequently, it is hard to distinguish between past HCV infection and biological false positivity with an anti-HCV result alone. We assessed the diagnostic performance of two immunoassays: the ARCHITECT anti-HCV chemiluminescent microparticle immunoassay (CMIA; Abbott Diagnostics, Wiesbaden, Germany) and the Access HCV Ab PLUS chemiluminescent immunoassay (CIA; Bio-Rad, Marnes-la-Coquette, France). We also explored an optimized algorithm to determine the anti-HCV results. METHODS: We tested 126,919 patients and 44,556 individuals who underwent a medical checkup. RIBA and NAAT were conducted for samples that tested anti-HCV-positive using CMIA and CIA. We assessed the optimal signal-to-cutoff (S/CO) ratio in HCV-positive samples. RESULTS: In total, 1,035 blood samples tested anti-HCV-positive. Of these, RIBA was positive in 512, indeterminate in 160, and negative in 363 samples. One hundred sixty-five samples were NAAT-positive. Diagnostic sensitivity and positive predictive value (PPV) were 96.7% and 52.1%, respectively, for CMIA, and 94.7% and 72.3%, respectively, for CIA. The optimal S/CO ratio was 5.2 for CMIA and 2.6 for CIA at 95% PPV. In total, 286 samples tested positive in CMIA and 444 in CIA, while 443 samples tested positive in both assays. CONCLUSIONS: It is hard to determine anti-HCV positivity based on the S/CO ratio alone. However, this study elucidated the role of the S/CO ratio by using the NAAT and RIBA.

Estudio Poblacional De Prevalencia De Cisticercosis Porcina En Atahualpa, Ecuador. Metodología Y Definiciones Operacionales / Population-Based Study Of Porcine Cysticercosis Prevalence In Atahualpa, Ecuador. Methodology And Operational Definitions

RESUMEN Objetivo: Evaluar la prevalencia de cisticercosis porcina en la parroquia rural Atahualpa, un poblado donde se ha demostrado endemia de neurocisticercosis humana. Diseño: Para el presente estudio, se colectarán muestras de sangre de aproximadamente 600 cerdos, las cuales serán procesadas mediante la prueba de EITB para estimar la tasa actual de sero-prevalencia de cisticercosis porcina. Unicamente se considerará el muestreo en cerdos a partir de los 2 meses de edad. Asimismo, solo se considerarán a aquellos animales sin evidencia de manifestaciones clínicas que hagan riesgosa su manipulación durante la toma de muestras de sangre. El presente estudio no contempla riesgo para la salud de los animales durante la toma de muestras. De acuerdo a las características de endemicidad de cisticercosis humana en Atahualpa, consideramos que la seroprevalencia esperada de cisticercosis porcina mediante EITB sea de al menos 25%. Entre los objetivos secundarios está el determinar los factores relacionados a una mayor o menor seroprevalencia de cisticercosis porcina (de la esperada). Todos los cerdos que resulten positivos (al menos 3 bandas) en el EITB, serán subsecuentemente tratados con oxfendazole, utilizando el régimen de dosis única de 30 mg/kg. Comentario: La importancia de definir la presencia de infección en la población porcina de una zona híper-endémica de neurocisticercosis humana radica en confirmar la existencia de transmisión activa. Si ésta es demostrada, el tratamiento de los cerdos infectados ayudará a interrumpir el ciclo de la Taenia solium y reducirá la prevalencia de esta enfermedad.

ABSTRACT Objective: To assess sero-prevalence of porcine cysticercosis in Atahualpa, a rural Ecuadorian village endemic for human neurocysticercosis. Design: We aim to collect at least 600 blood samples of local pigs, to quantify the number of positive bands in the EITB, and to estimate crude prevalence rates of porcine cysticercosis in the village. Sampling will only include pigs older than 2 months of age. Only healthy pigs will be sampled, to avoid risks inherent to manipulation of sick animals. There will be no risk for pigs as the result of the procedure. According to the estimated endemicity of human cysticercosis, we expect to find about 25% of infected pigs in this study. In the event of prevalence rates above or below the expected percentage, we will attempt to assess the causes of such discrepancies. All pig positive to 3 bands of more in the EITB will receive treatment with oxfendazole at a single dose of 30 mg/kg. Comment: The importance of defining the prevalence of porcine cysticercosis in a village endemic for human neurocysticercosis is to confirm the existence of active transmission. If this is true, treatment of infected pigs will help to interrupt the life cycle of Taenia solium, thus reducing the prevalence of this disease in humans.

Diagnóstico e fatores de risco do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais / Diagnosis and risk factors of bovine taeniasis-cysticercosis complex in Salinas, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)

Analysis of anti-nuclear antibody and anti-nuclear antibody spectrum in 2 325 patients / 国际检验医学杂志

Objective To investigate the clinical value of anti-nuclear antibody(ANA) and anti-nuclear antibody spectrum(ANAs) detection.Methods A total of 2 325 patients with or suspected with autoimmune diseases(AID) were enrolled and detected for ANA and ANAs by using indirect immunofluorescence assay(IIF) and linear immunoblot assay(LIA) respectively.All detected results were analyzed.Results Among 2 325 patients,896 cases(38.54%) were positive with ANA,with positive rate of 45.46% in female patients,which was higher than the 18.46% of male patients(P<0.05),and the common fluorescence patterns were nuclear particle pattern,nuclear homogeneous pattern and the nucleolus pattern.816 cases(35.10%) were positive with ANAs,and the positive rates of anti-Sjogren's syndrome(SS)-B antibody,anti Ro-52 antibody and anti SS-A antibody were relatively higher.The consistency rate of the two methods was 91.66%.Conclusion ANA and ANAs detection could be with certain correlation,but might be not completely consistent,detection could improve the detection rate and reduce the missed detection rate.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4–11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease β subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Perfil da transmissão e prevalência da cisticercose bovina em propriedades rurais do Triângulo Mineiro / Transmission and prevalence profile of bovine cysticercosis in rural properties of Triangulo Mineiro, Brazil

A cisticercose pode acometer o bovino, que desenvolve a larva da Taenia saginata. Apesar da importância da cisticercose para a saúde pública, para a saúde animal e para economia, a realidade epidemiológica da ocorrência dessa zoonose no Brasil é pouco conhecida. No estado de Minas Gerais a maioria dos dados é obtida dos serviços oficiais de Inspeção Sanitária nos matadouros-frigoríficos, sendo poucas as informações baseadas em dados de campo oriundos de animais vivos. Com o objetivo de avaliar a situação epidemiológica da cisticercose bovina em pequenas e médias propriedades rurais de quatro municípios, situados na região do Triângulo Mineiro, se pesquisou a ocorrência e os principais fatores de risco associados à transmissão da doença nessas propriedades. Dessa forma, foi realizado um estudo epidemiológico de corte transversal envolvendo as 68 propriedades localizadas nas zonas rurais dos quatro municípios, onde foram coletadas 1002 amostras de sangue bovino e aplicado um questionário epidemiológico. O diagnóstico sorológico da cisticercose foi realizado por triagem pelo teste ELISA indireto e os casos suspeitos foram submetidos ao Imunoblot para confirmação. Este estudo revelou a prevalência de 4,7% de cisticercose bovina nas propriedades da zona rural dos municípios amostrados, no período de julho a agosto de 2013. Os fatores que se mostraram estatisticamente significantes para a transmissão da cisticercose foram a expertiness do responsável pela propriedade (RC 5,81 IC 1,87-8,66), a renda familiar (RC 6,02 IC 1,33-8,77) e a qualidade da água da propriedade (RC 9,40 IC 1,97-9,59). Outros fatores foram observados com menor significância. Esses resultados identificaram os principais fatores que interferem na transmissão da cisticercose bovina, permitindo subsidiar medidas de prevenção e controle dessa importante zoonose na região do Triângulo Mineiro.(AU)

Cysticercosis can affect cattle which develop the larvae Taenia saginata. Despite the importance of cysticercosis for public animal health and for the economy, the epidemiological reality of such zoonosis in Brazil is little known. In the southeastern region of the State of Minas Gerais most of the data are obtained from the official Sanitary InspectionServices in slaughterhouses, but little information based on field data from live animals. In order to assess the epidemiological situation of bovine cysticercosis in small and medium-sized rural properties of four municipalities, located in the region of Triângulo Mineiro, we searched the occurrence and the major risk factors associated with transmission of the disease in those properties. We conducted a cross-sectional epidemiological study involving 68 properties located in rural areas of the four municipalities, where there were collected 1002 bovine blood samples and applied an epidemiological questionnaire. The serological diagnosis of cysticercosis was conducted by sorting through indirect ELISA test, and the suspected cases were subjected to Immunoblot for confirmation. This study revealed the prevalence of 4.7% of bovine cysticercosis in rural properties of the municipalities sampled from July to August 2013. The factors which were statistically significant for the transmission of cysticercosis were the expertise of those responsible for the property (OR 5.81 IC 1,87-8.66), the family income (OR 6.02 IC 1.33-8.77) and the water quality of the property (OR 9.40 IC 1.97-9.59). Other factors observed were of lower significance. These results identified the main factors which interfere in the transmission of bovine cysticercosis, allowing to subsidize preventive measures and to control this important zoonotic disease in the region of Triângulo Mineiro.(AU)

Intra-uterine exposure of horses to Sarcocystis spp. antigens / Exposição intrauterina ao Sarcocystis spp. em equinos

The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.

O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.

Consideration of Appropriateness of Application of Immunoblot Assay as a Reentry Test for HCV or HIV Screening Reactive Donors / 대한수혈학회지

BACKGROUND: Currently, serological assay, immunoblotting, and nucleic acid amplification test (NAT) are required as reentry tests for deferred donors with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) screening reactive result. However, immunoblotting must be performed even for serological nonreactive donors. In this study, the efficacy of immunoblot applications for serological nonreactive donors in donor reentry procedures was examined. METHODS: We analyzed non-qualified donors with immunoblot results from 2011 to 2015 in Korea and investigated reentry procedures related with HCV or HIV in other countries. RESULTS: Percentages of donors who could not be released due to immunoblot results even with serological nonreactive results were 54.2% (1,824/3,367) for HCV and 35.9% (4,300/11,964). In the case of 662 donors, their results were considered to be different using other assay kits or based on other criteria. In other countries, immunoblotting is not required as a donor reentry test. CONCLUSION: Indeterminate or reactive immunoblotting results in serological nonreactive donors were due to nonspecific reactions. It is not reasonable to apply immunoblotting to serological nonreactive donors. Therefore, we suggest that immunoblot assays be excluded from the reentry test.

Diagnóstico do complexo teníase-cisticercose bovina em São João Evangelista, Minas Gerais, Brasil / Diagnosis of Bovine Taeniasis-Cysticercosis Complex in São João Evangelista, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina em Minas Gerais, Brasil, foi selecionado o município de São João Evangelista, onde foram coletadas amostras de sangue de 339 bovinos em 15 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também o diagnóstico de teníase humana por meio de exame coproparasitológico dos habitantes das propriedades. Encontrou-se a prevalência de 4,1% para cisticercose bovina e a frequência de 2,94% para teníase humana. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa (P=0,042) entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Concluiu-se que a cisticercose bovina está presente no município de São João Evangelista, MG, em índices considerados endêmicos, sendo o consumo de carne malpassada e não inspecionada o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the state of Minas Gerais, Brazil, the city of São João Evangelista was selected, and blood samples were collected from 339 cattle in 15 randomly selected farms. A socioeconomic questionnaire was filled in each property for the analysis of the factors that favor the maintenance of the taeniasis-cysticercosis complex. Additionally, there was the diagnosis of human taeniasis verified by stool examinations of the properties' inhabitants. A prevalence of 4.1% for bovine cysticercosis and the frequency of 2.94% for human taeniasis were found. Among the risk factors, a statistically significant relation (p = 0.042) was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is broadly distributed in the city of São João Evangelista, with rates considered endemic, being the consumption of raw and not-inspected meat the main risk factors for the maintenance of complex taeniasis-cysticercosis, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.

Relevant peptides of Taenia crassiceps for the diagnosis of bovine cysticercosis by immunoblot / Peptídeos relevantes de Taenia crassiceps para o diagnóstico da cisticercose bovina pelo immunoblot

Given the limited knowledge about the diagnosis of bovine cysticercosis by immunoblot, the aim of this study was to assess the applicability of this test, identifying key peptides with diagnostic value. Immunoblot assays were performed using total larval antigen of Taenia crassiceps and 60 sera of positive bovines for cysticercosis (30 naturally and 30 experimentally infected with T. saginata eggs), 30 sera of negative bovines for cysticercosis and 30 sera of bovines with other diseases (fascioliasis, hydatidosis and tuberculosis). The peptides of greater diagnostic importance, in descending order of accuracy (%), were as follows: 6-8kDa (90.8%), 129-143kDa (74.2%), 99-105kDa (71.7%) and 14-19kDa (71.1%). Cross-reactions, due to fascioliasis and hydatidosis, were observed in the four intervals of peptides highlighted. The results demonstrate that the total antigen of T. crassiceps has peptides with a high diagnostic potential; therefore, the immunoblot is useful in the diagnosis of bovine cysticercosis.

Tendo em vista o conhecimento limitado sobre o diagnóstico da cisticercose bovina pelo immunoblot, o objetivo deste trabalho foi avaliar a aplicabilidade desse teste, identificando os principais peptídeos com valor diagnóstico. Foram realizados ensaios de immunoblot, utilizando antígeno total de larva de Taenia crassiceps e 60 soros de bovinos positivos para a cisticercose (30 naturalmente e 30 experimentalmente infectados com ovos de T. saginata), 30 soros de bovinos negativos para a cisticercose e 30 soros de bovinos com outras patologias (fasciolose, hidatidose e tuberculose). Os peptídeos de maior importância diagnóstica, em ordem decrescente de acurácia (%), foram os seguintes: 6-8 kDa (90,8%), 129-143 kDa (74,2%), 99-105 kDa (71,7%) e 14-19 kDa (71,1%). Reações cruzadas, em decorrência da fasciolose e hidatidose, foram observadas nos quatro intervalos de peptídeos ressaltados. Os resultados demonstram que o antígeno total de T. crassiceps possui peptídeos com alto potencial diagnóstico, sendo, portanto, o immunoblot útil no diagnóstico da cisticercose bovina.

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

A New IgG Immunoblot Kit for Diagnosis of Toxoplasmosis in Pregnant Women

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG(R) kit recently recommended as a confirmatory test (96.7% of concordance).

Anisakiose: soropositividade humana e desenvolvimento de um modelo experimental / Anisakiasis: human seropositivity and development of an experimental model

Existem evidências científicas de que várias espécies de peixes do litoral do Estado do Rio de Janeiro estão contaminadas com a larva nematoide Anisakis simplex. A infecção em humano, a sororeatividade e a doença ainda não são bem conhecidas em território brasileiro. Também os modelos de estudo sa anisakiose experimental atuais em camundongos tem utilizado como norma geral a introdução da forma larvária ou de seu extrato antigênico por via intraperitoneal... Foram observados sororeatividade para antígenos somáticos e para os secretores/excretores apresentando valores significativamente mais elevados quando comparados aos controles (p,0,001) quando analisados por ANOVA e pós teste de Tukey. A relação direta entre a reatividade no ELISA e a frequência da ingestão sem relação com sintomas alérgicos sugerem a existência de contato prévio com antígenos do Anisakis simplex, mas não podem atestar uma tendência alérgica no grupo analisado. As taxas de reatividade em puérperas com pequena ingestão reforça esse raciocínio. Em animais, através deste experimento foi possível demonstrar a efetividade da utilização da via gástrica para sensibilização de uma infecção intestinal por larva viva de Anisakis simplex em camundongos.

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

IgG Western Blot for Confirmatory Diagnosis of Equivocal Cases of Toxoplasmosis by EIA-IgG and Fluorescent Antibody Test

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.