Celiaquía: Aprovechar el poder diagnóstico de los anticuerpos

La celiaquía es un trastorno mediado por la respuesta inmune al gluten ingerido en individuos genéticamente susceptibles. La enfermedad celíaca afecta al 1 % de la población mundial, y su incidencia se ha incrementado sustancialmente en las últimas décadas. Sin embargo, aún la enfermedad celíaca es pobremente reconocida por la comunidad médica y por la población, tanto a nivel internacional, como nacional, muchos casos permanecen subdiagnosticados. Para mejorar el diagnóstico y manejo del paciente celíaco se recomienda el uso oportuno de la serología específica de la enfermedad celíaca. De los distintos anticuerpos asociados con la enfermedad celíaca, los anticuerpos anti-transglutaminasa tisular (anti-TGt IgA) representan la primera opción diagnóstica por su elevada sensibilidad y especificidad. La prueba de anti-TGt IgA no solo permite descartar de modo confiable la celiaquía, sino funciona como filtro para la selección de pacientes tributarios de biopsia intestinal para la confirmación diagnóstica. El desarrollo de la serología ha posibilitado la aplicación de nuevas estrategias diagnósticas que obvian la biopsia intestinal al menos en algunos grupos de pacientes.

Celiac disease is a disorder mediated by the immune response to ingested gluten in genetically susceptible individuals. Celiac disease affects 1% of the world population, and its incidence has increased substantially in recent decades. However, celiac disease is still poorly recognized by the medical community and by the population, both domestic and international, many cases remain underdiagnosed. Improving the diagnosis and management of the celiac patient, the timely use of specific serology for celiac disease is recommended. Different antibodies associated with celiac disease, however, anti-tissue transglutaminase antibodies (anti-TGt IgA) represent the first diagnostic option due to their high sensitivity and specificity. The anti-TGt IgA test not only constantly rules out celiac disease, but also functions as a filter for the selection of patients eligible for intestinal biopsy for diagnostic confirmation. The development of serology has enabled the use of new diagnostic strategies that avoid intestinal biopsy, at least in some groups of patients.

Métodos diagnósticos de laboratório para coronavirus 2019 n-cov / Laboratory diagnostic methods for coronavirus 2019 n-CoV / Métodos de diagnóstico laboratorial para coronavirus 2019 n-CoV

INTRODUCCIÓN: las múltiples y desconocidas características del coronavirus 2019-nCoV causante del síndrome respiratorio agudo y de millones de muertes en todo el mundo impulsó al desarrollo rápido de técnicas y kits de diagnóstico de laboratorio basados en la amplificación de secuencias virales o en la determinación de anticuerpos en sangre, sin embargo, tras su aplicación se generaron desacuerdos entre el resultado con la verdadera positividad o negatividad de la enfermedad. OBJETIVO: a través de la siguiente revisión se busca caracterizar y valorar el uso de las técnicas moleculares y serológicas, en función de factores como la sensibilidad y especificidad, tipo de espécimen y blanco de detección. METODOLOGIA: se emplearon las bases de datos Scopus y Web of Since obteniéndose 136 artículos de los cuales 25 cumplieron los criterios de inclusión como el desarrollo, validación clínica y estandarización de la técnica, así como nuevas metodologías propuestas. RESULTADOS: 17 elementos corresponden al diagnóstico de SARS-CoV-2 por técnicas moleculares y 8 por técnicas serológicas. El mayor número de publicaciones halladas provienen de China (5) y Estados Unidos (4), países de Europa (8) y Asia (6) mientras que, en América Latina se encontraron publicaciones procedentes de Brasil (1) y Ecuador (1). CONCLUSION: en relación a lo expuesto los ensayos moleculares destacan en su alta sensibilidad y especificidad y son usadas en cualquier etapa de la enfermedad. Las pruebas serológicas por su parte se recomiendan como complemento a las pruebas moleculares así como para monitoreo de la enfermedad en lugar de único marcador diagnóstico. (AU)

INTRODUCTION: the multiple and unknown characteristics of coronavirus 2019-nCoV causing acute respiratory syndrome and millions of deaths worldwide prompted the rapid development of laboratory diagnostic techniques and kits based on the amplification of viral sequences or the determination of antibodies in blood, however, after their application disagreements were generated between the result with the true positivity or negativity of the disease. OBJECTIVE: the following review seeks to characterize and evaluate the use of molecular and serological techniques, according to factors such as sensitivity and specificity, type of specimen and detection target. METHODOLOGY: the Scopus and Web of Since databases were used, obtaining 136 articles of which 25 met the inclusion criteria such as development, clinical validation and standardization of the technique, as well as new proposed methodologies. RESULTS: 17 items correspond to the diagnosis of SARS-CoV-2 by molecular techniques and 8 by serological techniques. The greatest number of publications found came from China (5) and the United States (4), European countries (8) and Asia (6) while, in Latin America, publications were found from Brazil (1) and Ecuador (1). CONCLUSIONS: in relation to the above, molecular assays stand out for their high sensitivity and specificity and are used at any stage of the disease. Serological tests are recommended as a complement to molecular tests as well as for disease monitoring instead of being the only diagnostic marker.(AU)

INTRODUÇÃO: as características múltiplas e desconhecidas do coronavírus 2019-nCoV, causando síndrome respiratória aguda e milhões de mortes em todo o mundo, levaram ao rápido desenvolvimento de técnicas e kits de diagnóstico laboratorial baseados na amplificação de sequências virais ou na determinação de anticorpos no sangue, porém, após sua aplicação foram geradas discordâncias entre o resultado com a verdadeira positividade ou negatividade da doença. OBJETIVO: a revisão a seguir visa caracterizar e avaliar o uso de técnicas moleculares e serológicas, dependendo de fatores como sensibilidade e especificidade, tipo de espécime e alvo de detecção. METODOLOGÍA: scopus e Web of Since foram utilizados, obtendo 136 artigos dos quais 25 preenchiam os critérios de inclusão, como desenvolvimento, validação clínica e padronização da técnica, assim como novas metodologias propostas. RESULTADOS: 17 itens correspondem ao diagnóstico do SARS-CoV-2 por técnicas moleculares e 8 por técnicas serológicas. O maior número de publicações encontradas veio da China (5) e dos Estados Unidos (4), países europeus (8) e da Ásia (6) enquanto, na América Latina, foram encontradas publicações do Brasil (1) e do Equador (1). CONCLUSÕES: em relação ao acima exposto, os ensaios moleculares se destacam por sua alta sensibilidade e especificidade e são utilizados em qualquer estágio da doença. Os testes serológicos são recomendados como um complemento aos testes moleculares, bem como para o monitoramento de doenças, em vez de serem o único marcador de diagnóstico.(AU)

Fluorescent polarization assay, two versions of indirect and competitive enzyme- linked immunoassay for brucellosis diagnosis in vaccinated buffaloes (Bubalus bubalis) / Teste de polarização fluorescente, duas versões de imunoensaio enzimático indireto e imunoensaio competitivo para diagnóstico de brucelose em búfalos vacinados (Bubalus bubalis)

ABSTRACT: The fluorescence polarization assay (FPA), two variants (V) of the indirect enzyme-linked immunosorbent assay (I-ELISA) and the competitive enzyme-linked immunosorbent assay (C-ELISA) were evaluated in buffaloes to detect antibodies against Brucella spp. The V1 of I-ELISA identifies them through the monoclonal (M23) anti-bovine IgG (I-ELISAM23) and the V2 through the ProteinA / G (I-ELISA-A/G). Serum samples of 862 buffaloes (Bubalus bubalis) from the Northeast of Argentina (NEA) were analyzed using the complement fixation test (CFT) as the reference. Receiving Operator Characteristic (ROC) analysis defined for the area under the curve (AUC) determined the cutoff points, sensitivity (Se) and specificity (Sp) for each test. CFT identified 107 positive and 755 negative sera. The best AUC (0.986), Concordance with CFT (96.3%) and kappa value (0.843) was obtained by I-ELISA A/G test. This assay showed the highest Se (95.33%) and C-ELISA the highest Sp (97%). FPA failed to measure the antibodies in 23 (2.65%) serum samples due to unsuccessful reading. I-ELISA M23 proved to be ineffective to diagnose brucellosis in bubaline sera. The four serological tests showed cutoff points lower than those standardized for bovines. As conclusion, I-ELISA A/G, C-ELISA and FPA with its limitations would be effective techniques for the diagnosis of brucellosis in buffaloes in the NEA, requiring an appropriate cut-off point to guarantee their maximum performance in this species.

RESUMO: O ensaio de polarização de fluorescência (FPA), duas variantes (V) do ensaio imunoenzimático indireto (I-ELISA) e o ensaio imunoenzimático competitivo (C-ELISA), foram avaliados em búfalos para detectar anticorpos contra Brucella spp. O V1 do I-ELISA os identifica através do IgG monoclonal (M23) anti-bovino (I-ELISAM23) e o V2 ​​através da Proteína A / G (I-ELISA-A / G). Amostras de soro de 862 búfalos (Bubalus bubalis) do Nordeste da Argentina (NEA) foram analisadas usando o teste de fixação do complemento (CFT) como referência. A análise Receiving Operator Characteristic (ROC) definida pela área sob a curva (AUC) determinou os pontos de corte, sensibilidade (Se) e especificidade (Sp) de cada teste. A CFT identificou 107 soros positivos e 755 soros negativos. Os melhores valores de AUC (0.986), concordância com CFT (96.3%) e kappa (0.843) foram obtidos pelo teste I-ELISA A / G. Este ensaio mostrou a maior Se (95.33%) e C-ELISA a maior Sp (97%). O FPA falhou em medir os anticorpos em 23 (2,65%) amostras de soro devido à falha na leitura. O I-ELISA M23 provou ser ineficaz para o diagnóstico de brucelose em soros bubalinos. Os quatro testes sorológicos mostraram pontos de corte inferiores aos padronizados para bovinos. Em conclusão, I-ELISA A / G, C-ELISA e FPA com suas limitações seriam técnicas eficazes para o diagnóstico de brucelose em búfalos no NEA, exigindo um ponto de corte adequado para garantir seu desempenho máximo nesta espécie.

Diagnóstico micológico por técnicas no convencionales en el siglo XXI / Mycological diagnosis by non-conventional techniques in the 21st century

Introducción: En la actualidad las infecciones fúngicas representan un problema para la salud humana. Las infecciones causadas por especies patógenas de hongos registran un incremento constante y se ubican entre el cuarto y décimo lugar como causa de muerte, particularmente en las unidades de cuidado intensivo. Un diagnóstico adecuado y precoz impacta directamente en la morbilidad y mortalidad asociadas a estas. Objetivo: Describir las principales técnicas de diagnóstico no convencional de las enfermedades fúngicas más frecuentes, en especial las relacionadas con el diagnóstico serológico y molecular. Métodos: Se realizó una revisión de la literatura científica sobre el tema, publicada entre 2000 y 2019. Se revisaron un total de 63 trabajos. Como motores de búsqueda se emplearon Google y Google Scholar. Se revisaron las bases de datos Medline, PubMed, Science Direct, BUCea y SciELO. Análisis y síntesis de la información: Las técnicas serológicas se emplean en el diagnóstico de las micosis invasivas o sistémicas por ser fáciles, rápidas y confiables. La detección de anticuerpos tiene utilidad limitada en el diagnóstico de las micosis invasivas debido a que la respuesta puede estar retrasada, reducida o no existir en pacientes inmunocomprometidos. La detección de componentes no antigénicos liberados por los hongos durante la infección y la secuenciación de ácidos nucleicos fúngicos son otras opciones para el diagnóstico de las micosis. Conclusiones: El desarrollo biotecnológico aporta nuevas herramientas que incrementan las oportunidades de identificación de las micosis. En la actualidad se disponen de métodos basados tanto en la detección de marcadores inmunológicos como de elementos moleculares específicos. La eficacia de las herramientas no convencionales para el diagnóstico depende de la correcta combinación de estas(AU)

Introduction: Fungal infections are a current human health problem. Infections caused by pathogenic fungal species constantly increase in number, and are ranked between the fourth and tenth leading causes of death, particularly in intensive care units. Early accurate diagnosis has a direct impact on the morbidity and mortality of fungal infections. Objective: Describe the main non-conventional diagnostic techniques for the most common fungal diseases, especially those related to serological and molecular diagnosis. Methods: A review was conducted of the scientific literature about the topic published between the years 2000 and 2019. A total 63 publications were reviewed. The search engines used were Google and Google Scholar. The databases Medline, PubMed, Science Direct, BUCea and SciELO were reviewed. Data analysis and synthesis: Serological techniques are used for the diagnosis of invasive or systemic mycoses because they are easy, fast and reliable. The detection of antibodies has a limited usefulness in invasive mycosis diagnosis, for the response may be delayed, reduced or inexistent in immunocompromised patients. Detection of non-antigenic components released by fungi during infection and sequencing of fungal nucleic acids are other mycosis diagnosis options. Conclusions: Biotechnological development contributes new tools increasing mycosis identification opportunities. Methods are currently available which are based on detection of immunological markers and specific molecular elements. The efficacy of non-conventional diagnostic tools depends on their appropriate combination(AU)

Seroprevalencia a Mycobacterium avium subsp. paratuberculosis en bovinos criollos colombianos Romosinuano y costeño con cuernos / Seroprevalence to Mycobacterium aviumsubsp. paratuberculosis in the colombian creole cattle Romosinuano and costeño con cuernos

RESUMEN Objetivo. Determinar la seroprevalencia y los factores epidemiológicos asociados a Mycobacterium avium subsp Paratuberculosis (MAP) en dos razas de bovinos criollos del centro de investigación AGROSAVIA-Turipaná. Materiales y métodos. Se realizó un estudio epidemiológico de corte transversal. Fueron muestreados 848 bovinos criollos, 403 Romosinuanos y 445 costeño con cuernos (CCC); para el diagnóstico serológico de anticuerpos se utilizó la prueba Elisa Indirecta mediante el kit comercial Parachek® de Prionics. Las variables sexo, edad, raza y tipo de hato fueron los factores epidemiológicos evaluados y correlacionados con la presencia de anticuerpos contra MAP; la asociación estadística fue determinada mediante Odds Ratio y con un modelo multivariado de regresión logística, utilizando un nivel de significancia con p<0.05. Resultados. La seroprevalencia general de los bovinos criollos a MAP fue de 2.35% (IC 95%, 1.34-3.38); sin embargo, en los Romosinuano fue de 0.74% y en los CCC fue de 3.82%, siendo las diferencias estadísticamente significativas (valor p=0.003). También, empleando un análisis univariado, fueron más afectados las hembras y los animales mayores a un año. El análisis multivariado identificó como factores epidemiológicos las variables raza y sexo. Conclusiones. En las razas criollas del centro de investigación AGROSAVIA-Turipaná, la seroprevalencia a MAP fue baja; sin embargo, en la raza CCC el riesgo de contraer la enfermedad es seis veces mayor con relación a la raza Romosinuano. Más aún, se pudo evidenciar que las hembras tienen mayor riesgo de adquirir la enfermedad.

ABSTRACT Objective. Determine the seroprevalence and epidemiological factors associated with Mycobacterium avium subsp. paratuberculosis (MAP) in two Creole cattle breeds of the Turipaná research center -AGROSAVIA. Materials and methods. A cross-sectional epidemiological study was conducted,a total of 848 Creole bovine animals were sampled, 403 Romosinuano and 445 costeño con cuernos (CCC); for the serological diagnosis of antibodies, the Elisa Indirect test was used with the commercial kit Parachek®2 by Prionics. The variables sex, age, breed and herd type were the epidemiological factors evaluated and correlated with the presence of antibodies against MAP; the statistical association was established using the Odds Ratio and a multivariate logistic regression model, employing a significance level with p<0.05. Results. The general seroprevalence of the Creole cattle to MAP was 2.35% (95% CI, 1.34-3.38); however, in the Romosinuano it was 0.74% and in the CCC it was 3.82%, being this difference statistically significant (p=0.003). Furthermore, employing a univariate way analysis, females and animals older than one year of age were more affected. The multivariate analysis identified the breed and sex variables as epidemiological factors. Conclusions. In the Creole breeds of the AGROSAVIA-Turipaná research center, MAP seroprevalence was low; however, in the Costeño Con Cuernos breed, the risk of contracting the disease is six times higher than in the Romosinuano breed. Moreover, it was shown that females have a higher risk of acquiring the disease.

Performance of the Dot-blot test method for detecting antibodies to Sarcocystis spp. in cattle / Desempenho do teste Dot-blot para detecção de anticorpos para Sarcocystis spp. em bovinos

Serological techniques can detect antibodies against Sarcocystis spp., Neospora caninum and Toxoplasma gondii antigens in single or mixed infections. Immunofluorescent antibody tests (IFAT) is considered the gold standard technique for Sarcocystosis diagnostic in cattle serum and a positive IFAT result reflects Sarcocystis spp. infection. Therefore, the aims of the present study were to compare IFAT and Dot-blot for sarcocystosis diagnostic in experimentally infected mice and to investigate serological cross-reactions with N. caninum and T. gondii in these methods. Mice (Mus musculus) were inoculated intraperitoneally with bradizoites of Sarcocystis spp. or tachyzoites of N. caninum or T. gondii. Serum samples were obtained and analyzed by IFAT and Dot-blot for the three protozoa. Serum from N. caninum and T. gondii experimentally infected mice were tested by IFAT and reacted only to N. caninum or T. gondii antigens, respectively. Specific antibodies against Sarcocystis spp. were present in all animals experimentally infected with this protozoan, with IFAT titers from 10 to 800. Serum samples from mice experimentally infected with Sarcocystis spp., N. caninum and T. gondii and tested by Dot-blot demonstrated no cross reaction between protozoa. A Dot-blot using Sarcocystis spp. antigen appears to be a good alternative to IFAT in the serological diagnosis of Sarcocystosis.(AU)

As técnicas sorológicas podem detectar anticorpos contra os antígenos de Sarcocystis spp., Neospora caninum e Toxoplasma gondii em infecções únicas ou mistas. O teste de anticorpos imunofluorescentes (IFAT) é considerado a técnica padrão-ouro para o diagnóstico de sarcocistose no soro de bovinos e um resultado positivo de IFAT reflete Sarcocystis spp. infecção. Portanto, os objetivos do presente estudo foram comparar IFAT e Dot-blot para diagnóstico de sarcocistose em camundongos infectados experimentalmente e investigar reações cruzadas sorológicas com N. caninum e T. gondii nesses métodos. Os camundongos (Mus musculus) foram inoculados intraperitonealmente com bradizoítos de Sarcocystis spp. ou taquizoítos de N. caninum ou T. gondii. As amostras de soro foram obtidas e analisadas por IFAT e Dot-blot para os três protozoários. O soro de N. caninum e T. gondii infectados experimentalmente foram testados por IFAT e reagiram apenas aos antígenos de N. caninum ou T. gondii, respectivamente. Anticorpos específicos contra Sarcocystis spp. estavam presentes em todos os animais experimentalmente infectados com este protozoário, com títulos de IFAT de 10 a 800. Amostras de soro de camundongos infectados experimentalmente com Sarcocystis spp., N. caninum e T. gondii e testadas por Dot-blot não demonstraram reação cruzada entre protozoários. Um Dot-blot usando Sarcocystis spp. O antígeno parece ser uma boa alternativa ao IFAT no diagnóstico sorológico da sarcocistose.(AU)

Zika virus serological diagnosis: commercial tests and monoclonal antibodies as tools

Zika virus (ZIKV), an emerging arthropod-borne virus (arbovirus) of the Flaviviridae family, is a current issue worldwide, particularly because of the congenital and neurological syndromes associated with infection by this virus. As the initial clinical symptoms of all diseases caused by this group are very similar, clinical diagnosis is difficult. Furthermore, laboratory diagnostic efforts have failed to identify specific and accurate tests for each virus of the Flaviviridae family due to the cross-reactivity of these viruses in serum samples. This situation has resulted in underreporting of the diseases caused by flaviviruses. However, many companies developed commercial diagnostic tests after the recent ZIKV outbreak. Moreover, health regulatory agencies have approved different commercial tests to extend the monitoring of ZIKV infections. Considering that a specific and sensitive diagnostic method for estimating risk and evaluating ZIKV propagation is still needed, this review aims to provide an update of the main commercially approved serological diagnostics test by the US Food and Drug Administration (FDA) and Brazilian National Health Surveillance Agency (ANVISA). Additionally, we present the technologies used for monoclonal antibody production as a tool for the development of diagnostic tests and applications of these antibodies in detecting ZIKV infections worldwide.(AU)

A descriptive study on prevalence pattern of Japanese encephalitis in State of Manipur

Background and Objective: Japanese encephalitis (JE) surveillance is not well established in many countries, and laboratory confirmation is challenging, the true extent and prevalence of the virus and burden of disease are not well understood. It is estimated that 67,900 clinical cases of JE occur annually despite the widespread availability of vaccine, with approximately 13,600–20,400 deaths and an overall incidence rate of 1.8/100,000 in the 24 countries with JE risk. The present study aimed at determining the prevalence rate (PR) and distribution (time, place and person) of JE cases in Manipur. This descriptive study was conducted over 24-month period (2016–2017). Materials and Methods: A total of 1770 cases of acute encephalitis syndrome tested for JE including 251 confirmed JE were diagnosed by IgM antibody-capture enzyme-linked immunosorbent assay. Results: The JE cases were most commonly reported in the age group of >15 years. Most of JE prevalence was seen in rural distribution in our study. There is a strong seasonal pattern of JE occurrence in Manipur which peaked in July–August and declined by October each year, which corresponds to the monsoon season. The JE cases were reported in all the districts of the state expanding in the plains and hill regions. Conclusions: The changing pattern of JE cases among different age groups was also observed in our study. The present study reveals the changing pattern of the prevalence of JE in the State of Manipur and initiated a systematic approach of JE surveillance also highlights the need for further expanding of surveillance across the state.

Avaliação de dois testes sorológicos comerciais para diagnóstico das infecções pelo FIV e pelo FeLV / Evaluation of two point-of-care tests to diagnosis of FIV and FeLV infections

FIV e FeLV são retrovírus associados principalmente com neoplasias. Dois testes rápidos são disponibilizados no Brasil para o diagnóstico dessas infecções: um kit de imunocromatografia de fluxo bidirecional (SNAP® Combo IDEXX) e um kit de imunocromatografia de fluxo lateral unidirecional (ALERE/BIONOTE Anigen Rapid). O objetivo deste estudo foi comparar o teste SNAP® com o teste ALERE. Amostras de sangue de 178 gatos foram testadas utilizando-se ambos os kits. A reação em cadeia de polimerase em tempo real (qPCR) foi empregada como método confirmatório para todos os resultados. O teste SNAP® apresentou sensibilidade e especificidade de 100% para FIV; a sensibilidade e a especificidade do teste ALERE foram de 96,15% e 98,68%, respectivamente. A sensibilidade e a especificidade para o FeLV foram de 93,02% e 96,30% para o teste SNAP® e de 90,70% e 97,78% para o teste ALERE. Ainda em relação ao FeLV, três amostras com resultado positivo na qPCR obtiveram resultado falso-negativo em ambos os testes. Não houve diferença estatisticamente significante entre os métodos. Considerando a qPCR como padrão-ouro, o teste SNAP® apresentou maior sensibilidade e especificidade para o FIV, e o teste ALERE apresentou maior especificidade para o FeLV. Os resultados mostraram uma boa correlação entre os testes.(AU)

FIV and FeLV are Retrovirus associated mainly with feline neoplasms. Two point-of-care tests are commercially available in Brazil for diagnosis of these infections: a bidirectional flow immunochromatography kit (IDEXX SNAP ® Combo) and a lateral unidirectional flow immunochromatography kit (ALERE/BIONOTE Anigen Rapid). The aim of this study was to compare SNAP ® and ALERE tests. Blood samples obtained from 178 cats were evaluated using both tests. Quantitative real-time polymerase chain reaction (qPCR) was used as confirmatory test for all samples. The sensitivity and specificity of SNAP ® test was 100% for FIV, and for ALERE test was 96.15% and 98.68%, respectively. The sensitivity and specificity for FeLV was 93.02% and 96.30% for SNAP ® test and 90.70% and 97.78% for ALERE test. Three samples with a qPCR positive result for FeLV obtained a false negative result in both SNAP ® and ALERE tests. There was no statistically significant difference between the two methods. Considering qPCR as gold standard method, the SNAP® test showed higher sensitivity and specificity for FIV, and the ALERE test presented higher specificity for FeLV. The results showed good agreement among the tests.(AU)

Preparation of multi-epitope recombinant diagnostic antigen of Mycobacterium tuberculosis / 生物工程学报

Multi-epitope recombinant diagnostic antigen (designated 'B102') of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni²⁺-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation, protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively. The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.

Design of a chimeric antigen containing multiple immunodominant epitopes and its use in detection of IgM antibodies against Rubella virus / 生物工程学报

A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.

Eukaryotic expression and serodiagnostic potential of HIV-1 p24 antigen / 中华微生物学和免疫学杂志

Objective To express HIV-1 capsid p24 antigen in an eukaryotic expression system and to evaluate its antigenicity and potential in the early diagnosis of HIV. Methods The full-length gene of HIV-1 p24 and the signal peptide DRVI gene were amplified by PCR. The signal peptide DRVI preceding the p24 gene was introduced using fusion PCR, and cloned into vector pDRVI1. 0. Two recombinant plas-mids pDRVI-p24 and pDRVI-p24s were constructed and transfected into 293F cells. Expression and secre-tion of p24 protein were detected by SDS-PAGE, Ni-NTA column chromatography and molecular sieve were used to purify p24s protein. The purified protein was identified by Western blot and indirect ELISA using hu-man/mouse HIV-1-positive serum samples. Results The eukaryotic expression system for HIV-1 p24 anti-gen was successfully established with high efficiency. The target protein of interest with the signal peptide DRVI was obviously detected in the supernatants of cell culture. The recombinant protein had good specifici-ty and sensitivity based on the results of serological tests using serum samples of five HIV-1-positive and five HIV-negative mice , 30 HIV-1-positive patients and 50 HIV-1-negative healthy individuals . Conclusions The eukaryotic expression system for HIV-1 p24 antigen was successfully established. The purified HIV-1 p24s antigen had good antigenicity. An indirect ELISA assay with good specificity and sensitivity for the de-tection of HIV-1 was preliminarily constructed and showed great potential for application.

Fatores associados à submissão ao teste rápido anti-HIV na assistência ao parto / Factors associated with submission to HIV rapid test in childbirth care

Resumo Os testes rápidos anti-HIV vêm sendo empregados nas maternidades com vistas à prevenção da transmissão vertical. O objetivo do estudo foi analisar os fatores associados à submissão ao teste rápido anti-HIV (desfecho). Estudo transversal, conduzido em 2009, em 15 hospitais do SUS do Rio de Janeiro/RJ, mediante entrevista a amostra representativa de 835 parturientes internadas e consulta a prontuários. Razões de prevalência ajustadas foram obtidas por regressão de Poisson, segundo modelo hierarquizado, permanecendo no modelo final as variáveis associadas ao desfecho (p ≤ 0,05). Segundo os prontuários (SP), 79,6% das mães foram submetidas ao teste rápido anti-HIV e, segundo as entrevistas (SE), 55,7%. No nível distal, a ausência de companheiro (SP), ter ≥ 6 moradores na residência (SP) e a cor da pele não branca (SE) se associaram a uma maior prevalência do desfecho. No nível intermediário, não dispor de sorologia negativa para o HIV do pré-natal (SP e SE) se associou a uma maior prevalência do desfecho, bem como a realização de pré-natal na rede básica (SP) e a não realização de pré-natal (SE). No nível proximal, o parto em hospital não certificado como amigo da criança se associou a uma maior prevalência do desfecho (SP e SE).

Abstract Rapid HIV tests are used in maternity hospitals to prevent mother-to-child transmission. This study aimed to analyze factors associated with submission to the rapid HIV test (outcome). This is a cross-sectional study conducted in 2009 in 15 hospitals from the Rio de Janeiro's Unified Health System (SUS) by interviewing a representative sample of 835 pregnant women hospitalized for birth and by verifying medical records. Adjusted prevalence ratios were obtained by Poisson regression according to a hierarchical model, and variables associated with the outcome (p ≤ 0.05) remained in the final model. According to medical records (MR), 79.6% of mothers were submitted to rapid HIV test and, according to interviews (INT), 55.7%. At the distal level, the lack of a partner (MR), having ≥ 6 residents at home (MR) and non-white skin color (INT) were associated with a higher prevalence of the outcome. At the intermediate level, not having a negative HIV serology from prenatal care (MR and INT) was associated with a higher prevalence of the outcome, as well as PHC prenatal care (MR) and lack of prenatal care (INT). At the proximal level, delivery in a hospital not certified as Baby-Friendly was associated with a higher prevalence of outcome (MR and INT).

Determinación del porcentaje de pacientes con enfermedad de chagas en la Amazonía Boliviana (parte i) / Determination of the percentage of patients with chagas disease in the Bolivian Amazon (part i)

OBJETIVO: Determinar la presencia de la enfermedad de Chagas en la Amazonía Boliviana mediante diagnóstico serológico y molecular en muestras de suero y sangre de pacientes de las regiones de Riberalta, Guayaramerín y Cobija. MATERIAL Y MÉTODOS: La población de estudio consistió en 192 personas mayores de edad habitantes de las regiones de Riberalta, Guayaramerín y Cobija quienes acudieron a los centros hospitalarios para una atención médica. Para el análisis de las muestras se utilizaron técnicas serológicas ELISA, HAI, IFI y la técnica molecular de la PCR en punto final. RESULTADOS: Los resultados reflejaron un porcentaje de infección del 4,08 por ciento del total de los pacientes de Cobija; 6,67 por ciento de los pacientes de Riberalta y 7,23 por ciento de los pacientes de Guayaramerín. Del total de los participantes el 74,48 por ciento fueron mujeres mayores de 17 años de edad. Los resultados de la PCR en punto final reflejaron un porcentaje de positividad general del 0,5 por ciento; los resultados de las pruebas serológicas reflejaron un porcentaje de positividad general del 6,25 por ciento.

OBJETIVE: To determine the presence of Chagas' disease in the Bolivian Amazon by serological and molecular diagnosis in serum and blood samples from patients from Riberalta, Guayaramerin and Cobija regions. MATERIAL AND METHODS: The study population was established by 192 elderly people living in the regions of Riberalta, Guayaramerin and Cobija who went to hospital for medical care. For the analysis of the samples serological techniques ELISA, HAI, IFI and the molecular technique of the PCR in end point were used. RESULTS: The results reflected a percentage of infection of 4,08 of the patients of Cobija, 6,67 percent of the patients of Riberalta and 7,23 percent of the patients of Guayaramerin. Of the total number of participants, 74,48 percent were women older than 17 years of age. The end-point PCR results showed a general positivity percentage of 0,5 percent the results of the serological tests reflected a general positive percentage of 6,25 percent.

Diseño de una prueba diagnóstica casera para la detección de anticuerpos tipo IgG contra el virus de la hepatitis C: incorporación de la proteína de envoltura E2 y no estructural 2 (NS2) / Design of an in-house diagnostic test for IgG antibodies against hepatitis C virus detection: Incorporation of envelope protein E2 and non-structural protein 2 ( NS2)

Durante la infección por el virus de la hepatitis C (VHC), los anticuerpos específicos aparecen varias semanas posterior a la exposición, van dirigidos contra las diversas proteínas del virus incluyendo anticuerpos contra la envoltura viral (E2)y la proteína no estructural 2 (NS2). En este trabajo se diseñó un ensayo casero de ELISA que incorpora, además de NS3, NS5a, NS5b y el core, a las proteínas E2 y NS2 del VHC en sustitución de NS4, con el fin de evaluar su capacidad diagnóstica en comparación con un estuche comercial de 4ta generación. La validación de la prueba casera demostró una especificidad y sensibilidad similar a las obtenidas con el estuche comercial de 4ta generación (Biokit©), con un índice kappa igual a 0,969, al compararse con el mismo. Esto sugiere que la prueba diseñada podría utilizarse de manera segura para la detección de anticuerpos VHC específicos de tipo IgG para el diagnóstico de la hepatitis C y constituirse como una alternativa de producción nacional más económica.

During hepatitis C (HCV) infection specific antibodies appear several weeks after exposure, including viral envelope (E2) and non-structural protein 2 ( NS2). In this work we designed an in-house ELISA assay, that incorporate beside NS3, NS5a, NS5b and core, the HCVE2 and NS2 proteins in substitution of NS4, in order to evaluate its diagnostic utility as compared to a fourth generation commercial kit. The in-house test demonstrated a specificity and sensitivity similar to those obtained with the commercial kit, with a kappa index equal to 0.969, when it was compared with the 4th generation commercial kit (BioelisaBiokit ©), suggesting that our test could be used for the diagnosis of specific IgG antibodies detection against hepatitis C and to become a more economic national alternative.

Current status of standards for laboratory diagnosis of pertussis and clinical relevance of serological diagnosis / 中华微生物学和免疫学杂志

Despite high coverage of vaccinations, incidence of pertussis (whooping cough) has been increasing throughout the world and large outbreaks were reported in several countries.Adolescents and adults with atypical pertussis symptoms have become the main sources of infants′ pertussis.Since clinicians are lack of experience in making diagnosis based on atypical symptoms, laboratory methods are needed.The currently recommended laboratory methods include bacterial culture, PCR and serology ELISA.It is well known that sensitivity and specificity of the above-mentioned methods may vary depending on many factors such as status of vaccination, timing of specimen collection and onset of symptoms.Serology ELISA to measure specific anti-pertussis toxin IgG antibodies in serum has been proven to be one suitable method for the diagnosis of pertussis infection in adolescents and adults.In this review, we summarize the current status of methods used in China and other countries for the laboratory diagnosis of pertussis and discuss the clinical relevance of serology ELISA for pertussis diagnosis.

Diagnostic value of serum Mp1p antigen detection for Talaromyces marneffei infection in acquired immune deficiency syndrome patients / 中华传染病杂志

Objective To explore the diagnostic value of Talaromyces marneffei (T.marneffei)-specific mannose glycoprotein Mp1p antigen for T.marneffei infection in acquired immune deficiency syndrome (AIDS) patients.Methods All cases were recruited in this study from January 2012 to June 2015 in Guangzhou No.8 People′s Hospital, including 184 AIDS patients with T.marneffei infection confirmatively diagnosed by culture, and 205 controls including 176 AIDS patients without T.marneffei infection and 29 health controls.Double antibody sandwich enzyme linked immunosorbent assay and fluoroimmunoassay combined with double-antibody sandwich were both utilized to detect serum Mp1p antigen levels, and their sensitivity and specificity for diagnosing T.marneffei infection in patients with AIDS were analyzed.x2 test and t test were used for statistical analysis.Results The ratio of males to females and age of the study group were both comparable to those of the control group (x2=0.019, P=0.889;t=1.810,P=0.07, respecitvley).The sensitivities of double antibody sandwich enzyme linked immunosorbent assay and fluoroimmunoassay combined with double-antibody sandwich were 82.07%(151/184) and 83.15%(153/184), respectively (x2=0.076, P=0.783).The specificities were 93.17%(191/205) and 92.68%(190/205), respectively (x2=0.037, P=0.847).The accuracy values were 87.92%(342/389) and 88.17%(343/389), respectively (x2=0.012, P=0.912).The false positive rates were 6.83%(14/205) and 7.32%(15/205), respectively.The false negative rates were 17.93%(33/184) and 16.85%(31/184), respectively (x2=0.049, P=0.829).The positive predictive values were 91.52%(151/165) and 91.07%(153/168), respectively (x2=0.021, P=0.886).The negative predictive values were 85.27%(191/224) and 85.97%(190/221), respectively (x2=0.045, P=0.832).The Kappa values were 0.83 and 0.80, respectively.Conclusion Detection of serum Mp1p antigen of T.marneffei possesses high specificity and sensitivity, which may be utilized for rapid and early diagnosis of T.marneffei infection in patients with AIDS.

Current status and research progress of serological antibody detection of Mycoplasma pneumonia / 国际儿科学杂志

Mycoplasma pneumonia(MP) is a common cause of children community-acquired pneumonia,and can lead to a variety of extrapulmonary complications.Thus,a rapid,sensitive diagnostic method is particularly important for early diagnosis and treatment.As MP grows slowly,requires additional nutrient supply and easy to be contaminated by fungi and bacteria during the culture process,culture of MP might not be suitable for clinical detection.However,the PCR technique is more complicated and high requirement for operator to be implemented in primary hospitals.Therefore,the serum diagnosis has become the commonly used method in clinical diagnosis.This paper is to review the latest findings of different serological diagnostic methods,antigen component,and antibody type in different periods of infection of MP.

Epidemiological survey of Neorickettsia risticii in equids from the State of Rio de Janeiro, Brazil / Levantamento epidemiológico de Neorickettsia risticii em equídeos do Estado do Rio de Janeiro

Equine neorickettsiosis (EN), also known as Potomac Horse Fever, is a non-contagious disease caused by the bacterium Neorickettsia risticii of the Anaplasmataceae family. The objectives of this study were to detect the presence of anti-N. risticii antibodies by the indirect immunofluorescence assay (IFA) and of its DNA by qPCR in equids at high and low altitude regions in the State of Rio de Janeiro, Brazil, and to identify factors associated with seropositive equids by multiple logistic regression analysis. The frequency of anti-N. risticii antibodies was 16.05% (n=113/704). The animal age and breeding region were the factors that influenced the seropositivity rate for N. risticii in the equids (p<0.05). Equids from the lowland region had higher seropositivity (p<0.05; OR=5.87) compared to those of the mountain region. The presence of snails on the farm was a factor associated with this result (p<0.05; OR=2.88). In the lowland region, age of the animal and site of breeding were protective factors for the detection of antibodies anti-N. risticii in equids, with lower frequency of seropositivity in younger animals (p<0.05; OR=0.06) and in animals raised in dry areas (p<0.05; OR=0.22). The presence of the target DNA of N. risticii by qPCR was not observed in any of the samples tested. The existence of seropositive equids for N. risticii demonstrates a possible circulation of this agent in the studied area, and that the age related characteristics and equids breeding region are important factors regarding seropositivity in the State of Rio de Janeiro.(AU)

A Neorickettisiose equina (NE), também conhecida como Febre do Cavalo de Potomac, é uma doença não contagiosa causada pela bactéria Neorickettsia risticii da família Anaplasmataceae. Os objetivos deste estudo foram detectar a presença de anticorpos anti-N. risticii através da reação de Imunofluorescência Indireta (RIFI) e do DNA dessa bactéria através da qPCR em equídeos de regiões de alta e baixa altitude no Estado do Rio de Janeiro, Brasil; e identificar os fatores associados com a soropositividade dos equídeos através da análise de regressão logística múltipla. A frequência de anticorpos anti-N. risticii foi de 16,05% (n=113/704). Observou-se que a idade e a região de criação foram os fatores que influenciaram a taxa de soropositividade para N. risticii nos equídeos (p<0,05). Equídeos da região de baixada apresentaram maior soropositividade (p<0,05; OR=5,87) quando comparado aos criados em região de montanha. A presença de caramujos na propriedade foi um fator associado a este resultado (p<0,05; OR=2,88). Na região de baixada, animais mais jovens (p<0,05; OR=0,06), criados em áreas secas (p<0,05; OR=0,22) demonstraram serem fatores de proteção na detecção de anticorpos anti-N. risticii. Não foi observada a presença do DNA-alvo de N. risticii através da qPCR em nenhuma das amostras testadas. A existência de equídeos soropositivos para N. risticii demonstra a possível circulação desse agente na área estudada, e as características inerentes a idade e a região de criação dos equídeos são fatores importantes relacionados à soropositividade no estado do Rio de Janeiro.(AU)

Aspectos soroepidemiológicos da leishmaniose visceral no Departamento Regional de Saúde (DRS) XV região de São José do Rio Preto, estado de São Paulo, Brasil, 2008-2012 / Seroepidemiological aspects of visceral leishmaniasis in the Regional Health Department (DRS) XV region of São José do Rio Preto, state of São Paulo, Brazil, 2008 - 2012

O objetivo deste estudo foi descrever as características soroepidemiológicas da leishmaniose visceral (LV) no DRS XV, região de São José do Rio Preto, São Paulo, entre 2008 e 2012. Foram analisados os dados secundários dos casos humanos, presença de vetores e resultados dos inquéritos sorológicos caninos. Desde os primeiros registros da LV na região em 2008, em Jales e Urânia, verificou-se crescente expansão atingindo outros 23 municípios até 2012. Foram notificados 251 casos suspeitos de LV humana (LVH), dos quais, 99 (39,4%) confirmados laboratorialmente, sendo 68 (68,7%) autóctones da região do DRS XV. Houve predomínio pelo sexo masculino, a faixa etária mais acometida foi para menores de 10 anos e maiores de 51 anos de idade...

The aim of this study was to describe the soroepidemiological characteristics of visceral leishmaniasis (VL) in DRS XV, region of São José do Rio Preto, São Paulo, between 2008 and 2012. The secondary data from human cases were analyzed, the presence of vectors and survey results serological canines. From the first records of VL in the region in 2008 in Jales and Urania, there was growing expansion reaching 23 other municipalities by 2012. Were reported 251 suspected cases of human VL (HVL), of which 99 (39.4%) laboratory confirmed, 68 (68.7%) of the autochthonous DRS XV region. There was a predominance of males, the most affected age group was for children under 10 and adults over 51 years old...