Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Inmunohistochemical detection of pandemic SARS-CoV-2 antigens in lung tissue / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARS- CoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Suplementação de probióticos: ferramenta no controle de infecções respiratórias virais ­ uma revisão sistemática / Probiotics supplementation: a tool in the control of viral respiratory infection ­ a systematic review

Infecções respiratórias virais estão entre as principais causas globais de adoecimento de acordo com o estado de saúde e o microbioma do indivíduo. O objetivo dessa revisão foi identificar possíveis efeitos associados à suplementação de probióticos em infecções respiratórias virais. Para tanto, realizou-se uma busca sistematizada nas bases de dados Google Acadêmico, Scopus e PubMed partindo da hipótese de que a intervenção clínica baseada na suplementação de probióticos reduz a gravidade dos sinais/ sintomas de infecções virais. Foram identificados 585 artigos dos quais foram selecionados 16 para compor a síntese descritiva deste artigo. O uso de probióticos como terapêutica na infecção respiratória tem capacidade de melhorar o quadro clínico do paciente por meio de: (i) modulação da resposta imune, (ii) melhora da resposta específica, (iii) produção de bacteriocinas, (iv) melhora na integridade de mucosas, (v) redução do número de cópias virais.

Viral respiratory infections are among the main global causes of illness according to the individual's health status and microbiome. The objective of this review was to identify possible effects associated with probiotic supplementation in viral respiratory infections. Therefore, a systematic search was carried out in the Google Academic, Scopus and PubMed databases, based on the hypothesis that clinical intervention based on supplementation of probiotics reduces the severity of signs/symptoms of viral infections. 585 articles were identified, of which 16 were selected to compose the descriptive synthesis of this article. The use of probiotics as therapeutics in respiratory infection is able to improve the patient's clinical condition through: (i) modulation of the immune response, (ii) improvement of the specific response, (iii) production of bacteriocins, (iv) improvement in mucosal integrity, (v) reduction in the number of viral copies.

Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients / Validação clínica de um ensaio de PCR in house quantitativo em tempo real para infecção por citomegalovírus usando o 1º Padrão Internacional da OMS em pacientes com transplantados renais

Abstract Introduction: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. Objective: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. Study design: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. Results: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. Conclusions: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

Resumo Introdução: Citomegalovírus (CMV) é um dos agentes infecciosos mais comuns em pacientes com transplante de órgãos sólidos, com morbidade e mortalidade significativas. Objetivo: Este estudo visou estabelecer um limite para o início do tratamento preemptivo. Além disso, comparou o desempenho da antigenemia com os resultados da qPCR in house. Desenho do estudo: Este foi um estudo de coorte prospectivo realizado em 2017 em um centro único de transplante renal no Brasil. A validação clínica foi realizada comparando resultados de qPCR in house, com o padrão de atendimento na época (Antigenemia para CMV Pp65). A análise da curva ROC foi realizada para determinar o limite ideal para o início da terapia preemptiva baseado nos resultados do teste qPCR in house. Resultados: 232 amostras de 30 pacientes foram testadas com antigenemia e qPCR, das quais 163 (70,26%) foram concordantes (Coeficiente Kappa: 0,435, p<0,001; Correlação Spearman: 0,663). PCR permitiu diagnósticos precoces. O número médio de dias para o primeiro resultado positivo foi 50 (intervalo, 24-105) para antigenemia e 42 (intervalo, 24-74) para qPCR (p<0,001). A análise da curva ROC revelou que em um limite de 3.430 UI/mL (Log 3,54), qPCR teve sensibilidade de 97,06% e especificidade de 74,24% (AUC 0,92617 ± 0,0185, p<0,001), na previsão de 10 células/10(5) leucócitos por antigenemia e na decisão do médico de tratar. Conclusões: Antigenemia para CMV Pp65 e qPCR para CMV mostraram uma concordância aceitável e uma correlação moderada neste estudo. qPCR in house revelou-se um método preciso para determinar DNAemia do CMV em pacientes transplantados renais, obtendo resultados positivos semanas antes da antigenemia.

Performance of an alternative RT-PCR procedure using residual sample from the Panbio™ Ag COVID-19 test

ABSTRACT Introduction: In the current standard of care (SoC) RT-PCR method for COVID-19, the patient's swab was extracted in viral transport media (VTM). For the PanbioTM COVID-19 Ag Rapid Test, the patient swab is flushed out in extraction buffer, of which a small fraction is used for testing, leaving more than half the sample unused. This study was designed to show that RT-PCR results from the residual sample of the PanbioTM COVID-19 Ag Rapid Test (called Novel RT-PCR) are not worse than the SoC RT-PCR result. Methods: The study was performed using (1) dilution series of five patient samples, and (2) 413 patient samples comparing SOC versus Novel RT-PCR results. Results: For the dilution series samples, all tested positive by both methods. The bias between Ct values of Novel RT-PCR and SoC RT-PCR did not exceed 3.00 Ct using primers N1 and N2. A total of 413 COVID symptomatic patients seeking COVID testing were tested, of which 89 patients tested positive and 324 tested negative with SoC RT-PCR. In 324 patients who tested negative with SoC RT-PCR, 323 tested negative with Novel RT-PCR, and one (1) tested positive. Out of 89 who tested positive with SoC RT-PCR, 80 tested positive with the Novel RT-PCR, and nine patients showed a negative test result. The Overall Percent Agreement for the 413 valid patient sample pairs was 97.5 [95% CI 97 to 98]. Conclusion: The study demonstrated that the performance of the Novel RT-PCR method is acceptable compared to the SoC RT-PCR method and can be a useful tool to perform RTPCR without the need for new swab collections.

Situación de enfermería en América, frente a la pandemia Covid-19 / NursingSituacion in America, in front of the Covid -19 pandemic

INTRODUCCIÓN: en el mismo año en que se declara Año Internacional de la Enfermería y Partería, la inesperada aparición del nuevo coronavirus SARS-CoV-2,dio un giro a lo que se tenía planeado dentro de los programas de salud a nivel mundial y deja en evidencia las debilidades de los sistemas sanitarios, donde el continente más afectado por dicho virus fue América, ya que sus esfuerzos por contener la pandemia fueron insuficientes, el tiempo de reacción para establecer protocolos de salud fue tardío y la disponibilidad para dotar al personal de salud de equipos de protección fue mínimo, y aun así el accionar del personal sanitario en especial de enfermería. OBJETIVO: describir la situación de enfermería en América, frente a la pandemia Covid-19. METODOLOGÍA: la investigación se realizó mediante un diseño narrativo, de carácter documental, analítico de enfoque cualitativo y método inductivo; obteniendo la información de fuentes secundarias confiables. RESULTADOS Y CONCLUSIONES: la actual pandemia demuestra la importancia de disponer de profesionales de salud en un número adecuado según las necesidades y cuidados que requiere cada paciente; es por esta razón que se precisa que los países inviertan en mejorar las condiciones de trabajo de los profesionales de enfermería, que incluyan equipos de protección individual, apoyo al trabajo en equipo y educación continua en enfermería, lo cual llevará a importantes logros, evidenciando el profesionalismo de enfermería y su entrega absoluta, al aplicar sus cuatro roles fundamentales con el fin de proteger la salud y mejorar la vida de las personas, a pesar de los evidentes riesgos reales y potenciales a los que se enfrentan a nivel laboral.

INTRODUCTION: in the same year in which the International Year of Nursing and Midwifery is declared, the unexpected appearance of the new SARS-CoV-2 coronavirus, gave a turn to what was planned within health programs worldwide and leaves in evidence the weaknesses of the health systems, where the continent most affected by this virus was America, since their efforts to contain the pandemic were insufficient, the reaction time to establish health protocols was late and the availability to provide staff with The health ofprotective equipment was minimal, and even so, the actions of health personnel, especially nursing personnel. OBJECTIVE: to describe the nursing situation in America, in the face of the Covid-19 pandemic. METHODOLOGY: the research was carried out through a narrative, documentary, analytical design with a qualitative approach and an inductive method; obtaining the information from reliable secondary sources. RESULTS AND CONCLUSIONS: the current pandemic shows the importance of having adequate numbers of health professionals according to the needs and care that each patient requires; It is for this reason that it is necessary for countries to invest in improving the working conditions of nursing professionals, which include individual protection equipment, support for teamwork and continuing education in nursing, which will lead to important achievements, evidencing the Nursing professionalism and its absolute dedication, by applying its four fundamental roles in order to protect health and improve people's lives, despite the obvious real and potential risks they face at the work level.

SARS-CoV-2 diagnostic testing alternatives for Latin America / Alternativas diagnósticas para SARS-CoV-2 para América Latina

Abstract In the past four months SARS-CoV-2 has reached most countries in the world. Public health strategies based on widespread testing and proper isolation of positive cases have shown to be helpful to reduce local transmission of SARS-CoV-2. Confirmatory tests, that identify viral RNA, and screening serological tests that identify viral antigens or host antibodies against viral proteins are part of the tools that nations can use to fight infectious disease epidemics. Understanding how each test works can provide insights about their test characteristics and how they can be used for different clinical and public health goals. Testing is a key strategy to reduce viral transmission, not only for this epidemic, but also for others to come.

Resumen En los últimos cuatro meses, el virus SARS-CoV-2 ha llegado a la mayoría de países en el mundo. Las estrategias de salud pública basadas en la realización masiva de pruebas diagnósticas y el aislamiento focalizado de casos positivos han demostrado ser útiles para la reducción de la transmisión de SARS-CoV-2. Las pruebas confirmatorias, que identifican el ARN viral, y las pruebas serológicas que identifican antígenos virales o anticuerpos contra las proteínas virales del huésped son herramientas que las naciones pueden usar para combatir las epidemias producidas por agentes infecciosos. El comprender cómo funcionan estas pruebas puede ayudar a entender sus características y cómo pueden ser usadas para diferentes objetivos clínicos y de salud pública. Las pruebas diagnósticas son herramientas clave para reducir la transmisión viral, no solo en esta epidemia, sino para otras por venir.

Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Natural versus recombinant viral antigens in SARS-COV-2 serology: challenges in optimizing laboratory diagnosis of COVID19

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in renal tissue in severe dengue in humans: Effects on endothelial activation/dysfunction

Abstract INTRODUCTION: Dengue is an important mosquito-borne disease in tropical and subtropical regions. Adhesion molecules have not been systematically characterized in the renal tissue of patients with severe dengue (SD). The objective of this study was to detect viral antigens in samples from patients that evolved with SD, correlating with the expression of ICAM-1, VCAM-1, VE-cadherin, and E-selectin to contribute to a better understanding of the pathophysiology of SD. METHODS: Kidney specimens from patients with SD were selected according to clinical and laboratorial data and submitted to histological and immunohistochemistry analysis. A semiquantitative evaluation was performed considering positive immunostaining in 20 glomeruli. RESULTS: Viral antigens were mainly detected in distal tubules. The intense immunostaining of VCAM-1 and ICAM-1 was observed. The expression of E-selectin was discrete, and VE-cadherin expression varied from mild to moderate. VCAM-1 was slightly intense in the glomerular capsule; the expression of ICAM-1 was diffuse. E-selectin was diffuse, and VE-cadherin varied from mild to moderate. The most frequent histological findings were glomerular congestion, mild glomerulitis, acute renal injury, and glomerular atrophy. CONCLUSIONS: The results appear to demonstrate an imbalance between vascular endothelial permeability regulating events in renal lesions in SD. The increase in the expression of ICAM-1 and VCAM-1 is an in-situ indicator of higher permeability with a consequent influx of cells favoring the inflammation of the endothelium. These molecules are important in the pathophysiology of the disease and provide the possibility of developing new markers for the evaluation, clinical follow-up, and therapeutic response of patients with SD.

Standardization of antigenemia and qpcr cut-off values in whole blood for the detection of cytomegalovirus disease in hiv patients

Abstract INTRODUCTION: We defined the cut-off values of the antigenemia and cytomegalovirus (CMV) DNA tests in HIV/AIDS patients to identify CMV disease. METHODS: A total of 97 samples from 68 patients with and without CMV disease were analyzed by viral DNA detection and antigenemia assay. RESULTS: Qualitative and quantitative results significantly differed between assays. The cut-off values for the antigenemia and qPCR assays were 1.5 positive cells/200,000 leukocytes and 3.715 log/mL, respectively. CONCLUSIONS: Antigenemia and qPCR are suitable for monitoring CMV disease in HIV patients, however, the threshold values should be determined within the centers where the patients are monitored.

Polyspecific antibodies without persisting antigen in multiple sclerosis, neurolupus and Guillain-Barré syndrome: immune network connectivity in chronic diseases / Anticorpos poliespecíficos sem antígeno persistente em esclerose múltipla, neurolupus e síndrome de Guillain-Barré: conectividade imunológica em doenças crônicas

ABSTRACT The polyspecific antibody synthesis in multiple sclerosis (MS) gained diagnostic relevance with the frequent combination of measles-, rubella- and varicella zoster antibodies (MRZ-antibody reaction) but their pathophysiological role remains unknown. This review connects the data for intrathecal polyspecific antibody synthesis in MS and neurolupus with observations in the blood of patients with Guillain-Barré syndrome (GBS). Simultaneously increased antibody and autoantibody titers in GBS blood samples indicate that the polyspecific antibodies are based on a general property of an immune network, supported by the deterministic day-to-day concentration variation of antibodies in normal blood. Strongly correlated measles- and rubella- antibody variations point to a particular connectivity between the MRZ antibodies. The immune network, which provides serological memory in the absence of an antigen, implements the continuous change of the MRZ pattern in blood, not followed by the earlier immigrated B cells without corresponding connectivity in the brain. This may explain the different antibody patterns in cerebrospinal fluid, aqueous humor and blood of the individual MS patient. A complexity approach must implement a different view on causation in chronic diseases and causal therapies.

RESUMO A síntese de anticorpos poliespecíficos em esclerose múltipla (EM) ganhou relevância diagnóstica com a combinação frequente de anticorpos contra sarampo, rubéola e varicela-zoster (reação de anticorpos MRZ), mas seu papel fisiopatológico permanece desconhecido. Esta revisão relaciona os dados da síntese intratecal de anticorpos poliespecíficos em EM e Neurolupus com observações no sangue de pacientes com síndrome de Guillain Barré (SGB). Simultaneamente, os títulos aumentados de anticorpos e autoanticorpos em amostras de sangue de SGB indicam que os anticorpos poliespecíficos se baseiam numa propriedade geral de uma rede imunitária, suportada pela variação determinística da concentração diária de anticorpos no sangue normal. As variações fortemente correlacionadas de anticorpos contra sarampo e rubéola apontam para uma conectividade particular entre os anticorpos MRZ. A rede imunitária, que fornece memória sorológica na ausência de um antígeno, implementa a mudança contínua do padrão MRZ no sangue, não seguida pelas células B que imigraram anteriormente sem conectividade no cérebro. Isto pode explicar os diferentes padrões de anticorpos no LCR, humor aquoso e sangue do paciente individual de EM. Uma abordagem complexa deve implementar uma visão diferente sobre a causalidade em doenças crônicas e terapias causais.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool / Teste rápido de detecção de antígenos para o diagnóstico do Vírus Sincicial Respiratório como ferramenta de diagnóstico

Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

Resumo Objetivo: Avaliar o teste QuickVue® RSV Test Kit (QUIDEL Corp, CA, EUA) para o diagnóstico rápido do vírus sincicial respiratório em crianças com doença respiratória aguda, comparandoo com a imunofluorescência indireta como padrão ouro. Visto que, no Brasil, testes rápidos para detecção de antígenos para vírus sincicial respiratório não são rotineiramente utilizados como ferramenta de diagnóstico, exceto para Dengue e Influenza. Métodos: Um total de 486 amostras de aspirado de nasofaringe de crianças menores de 5 anos com doença respiratória aguda, coletadas entre dezembro de 2013 e agosto de 2014, foram analisadas por imunofluorescência e pelo teste QuickVue®. Amostras com resultados discordantes entre os métodos foram submetidas a PCR em tempo real e sequenciamento. Resultados: Das 313 amostras positivas por IFI, 282 foram positivas no teste rápido (90%), 2 amostras foram positivas apenas no teste rápido (0.6%), 33 apenas na imunofluorescência (10.5%) e 171 foram negativas em ambos os métodos. As 35 amostras com resultados discordantes foram testadas por PCR em tempo real, sendo que duas que foram positivas apenas no teste rápido e 5 apenas na imunofluorescência confirmaram-se positivas. Não houve relação entre a ausência de positividade no teste QuickVue® com a carga ou com a cepa viral. O teste QuickVue® mostrou sensibilidade de 90.1%, especificidade 98.9%, valor preditivo positivo 99.3%, valor preditivo negativo de 94.6%, acurácia de 93.2% e índice de concordância de 0.85 em comparação à imunofluorescência. Conclusões: Nosso estudo demonstrou que o teste QuickVue® RSV pode ser efetivo na detecção precoce do vírus sincicial respiratório em amostras de aspirado de nasofaringe e é confiável como uma ferramenta de diagnósticos em pediatria.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Búsqueda de flavivirus en individuos con sospecha clínica de dengue y resultado negativo para el antígeno NS1 en Paraguay / Search of flavivirus in individuals with clinical suspicion of dengue and negative for the NS1 antigen in Paraguay

Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.

Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.