Uso da citometria de fluxo no diagnóstico sorológico da COVID-19 / The use of flow cytometry for the serological diagnosis of COVID-19

A pandemia da COVID-19 tem tido um impacto devastador em todo o mundo e levou ao rápido desenvolvimento de testes diagnósticos. Diferentes tecnologias vêm sendo utilizadas para a detecção de imunoglobulinas frente à infecção por SARS-CoV-2. Ensaios imunoenzimáticos (ELISA), quimioluminescentes e imunocromatográficos estão disponíveis e, no geral, apresentam poder diagnóstico limitado, principalmente para a detecção de IgA. A citometria de fluxo tem surgido como alternativa para o desenvolvimento de métodos sensíveis e específicos para a COVID-19 aplicados para diagnóstico, triagem e estratificação da doença. A citometria de fluxo é uma tecnologia óptica baseada em laser que detecta características físico-químicas de células ou partículas em um fluido heterogêneo. O artigo explora a citometria de fluxo para o diagnóstico da COVID-19 em duas estratégias para a detecção de anticorpos no soro ou plasma, uma utilizando antígenos virais expressos na superfície de células de mamíferos e outra com estes elementos imobilizados em microesferas (beads). A possibilidade de detecção rápida de múltiplos anticorpos simultaneamente, com pequeno volume de amostra e elevada sensibilidade e especificidade, torna a citometria de fluxo uma metodologia promissora para o laboratório clínico, como ferramenta de referência para auxiliar na contenção do processo pandêmico da COVID-19 e futuros eventos similares.

The COVID-19 pandemic has had a devastating impact around the world and has led to the rapid development of diagnostic tests. Different technologies have been used to detect immunoglobulins produced against SARS-CoV-2 infection. Immunoenzymatic (ELISA), chemiluminescent and immunochromatographic assays are available and, in general, they have limited diagnostic accuracy, especially for the detection of IgA. Flow cytometry has emerged as an alternative for the development of sensitive and specific methods for COVID-19 applied for diagnosis, screening and stratification of the disease. Flow cytometry is a laser-based optical technology that detects physicochemical characteristics of cells or particles in a heterogeneous fluid. The article explores flow cytometry for the diagnosis of COVID-19 in two strategies for detecting antibodies in serum or plasma, the first one using viral antigens expressed on the surface of mammalian cells and the other one with these elements immobilized on microspheres (beads). The possibility of rapid detection of multiple antibodies simultaneously, with a small sample volume and high sensitivity and specificity, makes flow cytometry a promising methodology for the clinical laboratory, as a reference tool to help stop the COVID-19 pandemic process and similar future events.

Evaluation of 1, 5 -Anhydroglucitol as a Biomarker for Type 2 Diabetes Mellitus in Patients without Overt Nephropathy

1,5-Anhydroglucitol (1,5-AG) is a non-fasting glycemic marker that responds to hyperglycemia excursions. The reduction in serum levels of 1,5-AG is associated with an increase in postprandial glycemia and glycosuria, phenomena that increase the risk and severity of diabetic complications. The objective is to assess the ability of 1,5-AG to discriminate type 2 diabetes (T2D) patients without overt kidney disease, for screening or diagnostic purposes. The Human Research Ethics Committee of Universidade Federal do Paraná (UFPR) approved the project. Serum samples from 567 individuals classified as healthy subjects (n = 291) and T2D (n = 276) with moderate glycemic control (HbA1c of 7-8%), matched by gender, were analyzed. Serum 1,5-AG levels were measured using an automated enzymatic method (GlycoMark, Inc.). Receiver Operating Characteristic (ROC) curve analysis for 1,5-AG showed sensibility of 65.3% and specificity of 91.1% to detect T2D at cut-off point of 92 µmol/L. The results were similar to the groups' discrimination by glycemia (sensibility/specificity, 62.2%; 89.0%) at cut-off point of 6.3 mmol/L. HbA1c was the best discriminator (sensibility/specificity, 87.4%; 94.2%) at a cut-off point of 5.8% (40 mmol/mol). The serum 1,5-AG concentration was not able to discriminate T2D in the presence of moderate glycemic control with no overt nephropathy.

Caracterização dos distúrbios da regulação ácido-base: uma abordagem didática e intuitiva / Characterization of acid-base regulation disorders: a didactic and intuitive approach

Em humanos, o pH sanguíneo é mantido em uma faixa estreita, entre 7,35 e 7,45. Diferentes mecanismos bioquímicos, de forma harmônica, atuam para a manutenção do pH fisiológico. Múltiplos processos patológicos podem promover alterações no pH e nos gases sanguíneos, caracterizando acidose (pH <7,35) ou alcalose (pH >7,45). A ruptura da homeostasia do pH é identificada pela medição do pH, pressão parcial de dióxido de carbono (pCO2), concentração do bicarbonato (HCO3-) e, adicionalmente, com a pressão de oxigênio (pO2) em sangue arterial, processo descrito como gasometria arterial. Este artigo revisa os principais elementos associados a compreensão das alterações e tem como objetivo central apresentar uma abordagem didática e intuitiva para a caracterização destes distúrbios; e também comenta sobre ferramentais digitais destinadas a interpretações das alterações da gasometria arterial que também são abordados, como programas para computadores em ambiente web e aplicativos para telefonia móvel.

In humans, blood pH is kept in a narrow range, between 7.35 to 7.45. Different biochemical mechanisms, in a harmonic way, act to maintain the physiological pH. Multiple pathological processes can promote changes in pH and blood gases, characterizing acidosis (pH <7.35) or alkalosis (pH> 7.45). The rupture of pH homeostasis is identified by measuring pH, partial pressure of carbon dioxide (pCO2), bicarbonate concentration (HCO3 - and, in addition, with the pressure of oxygen (pO2) in arterial blood, a process described as gasometry arterial. This article reviews the main elements associated with the understanding of acid-base changes and aims to present a didactic and intuitive approach to the characterization of these disorders; and also comments on digital tools for the interpretation of alterations in arterial blood gases are also covered, such as programs for computers in a web environment and applications for mobile phone.

COVID-19 e Diabetes: a relação entre duas pandemias distintas / COVID-19 and Diabetes: two distinct pandemics and their relationship

O mundo enfrenta duas pandemias distintas, mas que guardam alguma relação entre si. O Diabetes mellitus (DM) promove um estado crônico inflamatório que torna os afetados mais propensos a infecções em geral. A doença aguda causada pelo novo coronavírus, COVID-19, assim como o DM, altera o sistema imunológico, podendo ativar uma tempestade de citocinas deletéria ao hospedeiro. O DM tem sido considerado como um fator de risco independente da idade para a gravidade da COVID-19. De fato, pessoas com DM são propensas a um curso clínico de maior severidade da COVID-19 com maior taxa de morbimortalidade. Uma forte relação entre a COVID-19 e o DM reside no fato de que o SARS-CoV-2 utiliza a proteína ACE-2 como receptor para entrar na célula humana, a qual é superexpressa pelas células das ilhotas pancreáticas e ainda mais em pessoas com DM. Depois de invadir a célula do hospedeiro, o vírus degrada a ACE-2, reduzindo sua atividade anti-inflamatória. Somado a isso, acredita-se que o SARS-CoV-2 afete diretamente a parte endócrina do pâncreas, com consequente hiperglicemia. Entretanto, ainda não está claro de que forma as alterações glicêmicas podem estar relacionadas com a severidade da COVID-19; e se o SARS-CoV-2 tem potencial diabetogênico. Todavia, os mecanismos propostos para explicar a associação observada entre DM e a COVID-19 incluem inflamação, alterações na resposta imune, na coagulação e a agressão direta do vírus às células b pancreáticas. Como o diabetes está associado às manifestações graves da COVID-19, o primeiro passo é evitar a contaminação de pessoas com DM pelo SARS-CoV-2. Depois, todo paciente com COVID-19 que tenha DM deve ser considerado grave. E, por fim, a monitoração glicêmica frequente em pacientes com COVID-19 deve ser considerada, uma vez que o controle da hiperglicemia tem se mostrado eficaz na promoção de melhores desfechos clínicos.

The world faces two distinct pandemics, which have some relationship with each other. Diabetes mellitus (DM) promotes a chronic inflammatory state that makes the affected more prone to general infections. The acute disease caused by the new coronavirus, COVID19, as well as DM, alters the immune system, which can activate a harmful cytokine storm in the host. DM has been considered as an age-independent risk factor for the severity of COVID-19. In fact, people with DM are prone to a more severe clinical course of COVID19 with a higher rate of morbidity and mortality. A strong relationship between COVID-19 and DM lies in the fact that SARS-CoV-2 uses the ACE-2 protein as a receptor to enter the human cell, which is overexpressed by pancreatic islet cells, especially in people with DM. After invading the host cell, the virus degrades ACE-2, reducing its anti-inflammatory activity. In addition, SARS-CoV-2 is believed to directly affect the endocrine part of the pancreas, with consequent hyperglycemia. However, it is not clear how glycemic changes may be related to the severity of COVID-19; and, whether SARS-CoV-2 has diabetogenic potential. However, the mechanisms proposed to explain the observed association between DM and COVID-19 include inflammation, changes in the immune response, coagulation and direct aggression of the virus to pancreatic beta cells. As diabetes is associated with severe manifestations of COVID-19, the first step is to avoid contamination of people with DM by SARS-CoV-2. Then, every patient with COVID-19 who has DM should be considered severe. Finally, frequent glycemic monitoring in patients with COVID-19 should be considered, since the control of hyperglycemia has been shown to be effective in promoting better clinical outcomes

d-GDM: A mobile diagnostic decision support system for gestational diabetes

ABSTRACT Objective The aim of the study is to describe a portable and convenient software to facilitate the diagnostics of gestational (GDM) and pre-gestational diabetes (PGDM). Materials and methods An open source software, d-GDM, was developed in Java. The integrated development environment Android Studio was used as the Android operational system. The software for GDM diagnosis uses the criteria endorsed by the International Association of Diabetes and Pregnancy Study Group, modified by the World Health Organization. Results GDM diagnosis criteria is not simple to follow, therefore, errors or inconsistencies in diagnosis are expected and could delay the appropriate treatment. The d-GDM, was developed to assist GDM diagnosis with precision and consistency diagnostic reports. The open source software can be manipulated conveniently. The operator requires information regarding the gestational period and selects the appropriate glycaemic marker options from the menu. During operation, pressing the button "diagnosticar" on the screen will present the diagnosis and information for the follow up. d-GDM is available in Portuguese or English and can be downloaded from the Google PlayStore. A responsive web version of d-GDM is also available. The usefulness and accuracy of d-GDM was verify by field tests involving 22 subjects and 5 mobile phone brands. The approval regards user-friendliness and efficiency were 95% or higher. The GDM diagnosis were 100% correct, in this pilot test. d-GDM is a user-friendly, free software for diagnosis that was developed for mobile devices. It has the potential to contribute and facilitate the diagnosis of gestational diabetes for healthcare professionals.

Biomarkers of cardio-renal syndrome in uremic myocardiopathy animal model / Biomarcadores de síndrome cardiorrenal em modelo animal de miocardiopatia urêmica

ABSTRACT Introduction: Cardio-renal syndrome subtype 4 (CRS4) is a condition of primary chronic kidney disease that leads to reduction of cardiac function, ventricular hypertrophy, and risk of cardiovascular events. Objective: Our aim was to understand the mechanisms involved on the onset of CRS4. Methods: We used the nephrectomy 5/6 (CKD) animal model and compared to control (SHAM). Serum biomarkers were analyzed at baseline, 4, and 8 weeks. After euthanasia, histology and immunohistochemistry were performed in the myocardium. Results: Troponin I (TnI) was increased at 4 weeks (W) and 8W, but nt-proBNP showed no difference. The greater diameter of cardiomyocytes indicated left ventricular hypertrophy and the highest levels of TNF-α were found at 4W declining in 8W while fibrosis was more intense in 8W. Angiotensin expression showed an increase at 8W. Conclusions: TnI seems to reflect cardiac injury as a consequence of the CKD however nt-proBNP did not change because it reflects stretching. TNF-α characterized an inflammatory peak and fibrosis increased over time in a process connecting heart and kidneys. The angiotensin showed increased activity of the renin-angiotensin axis and corroborates the hypothesis that the inflammatory process and its involvement with CRS4. Therefore, this animal study reinforces the need for renin-angiotensin blockade strategies and the control of CKD to avoid the development of CRS4.

RESUMO Introdução: A síndrome cardiorrenal (SCR) tipo 4 é uma afecção da doença renal crônica primária que leva a redução da função cardíaca, hipertrofia ventricular e risco de eventos cardiovasculares. Objetivo: O objetivo do presente estudo foi compreender os mecanismos envolvidos no surgimento da SCR tipo 4. Métodos: Um modelo animal de nefrectomia 5/6 (DRC) foi comparado a animais de controle (Placebo). Biomarcadores séricos foram analisados no início do estudo e com quatro e oito semanas de estudo. Após eutanásia, foram realizados exames histológicos e de imunoistoquímica no tecido miocárdico. Resultados: Troponina I (TnI) estava aumentada nas semanas quatro (S4) e oito (S8), mas o NT-proBNP não apresentou diferenças. O diâmetro maior dos cardiomiócitos indicava hipertrofia ventricular esquerda. Os níveis mais elevados de TNF-α foram identificados na S4 com redução na S8, enquanto fibrose foi mais intensa na S8. A expressão de angiotensina mostrou elevação na S8. Conclusões: TnI parece sugerir lesões cardíacas em consequência da DRC, porém o NT-proBNP não sofreu alterações por refletir alongamento. O TNF-α evidenciou um pico inflamatório e a fibrose aumentou ao longo do tempo devido ao processo de conexão entre rins e coração. A angiotensina mostrou aumento da atividade do eixo renina-angiotensina, corroborando a hipótese do processo inflamatório e seu envolvimento com SCR tipo 4. Portanto, o presente estudo em modelo animal reforça a necessidade de em adotar estratégias com bloqueadores de renina-angiotensina e controle da DRC para evitar o desenvolvimento de SCR tipo 4.

Serum Fluorescent Advanced Glycation End (F-AGE) products in gestational diabetes patients

ABSTRACT Objectives Advanced glycation end products (AGEs) are involved in the pathogenesis and complications of diabetes mellitus (DM). Gestational DM (GDM) is characterized by increased glycemia and oxidative stress, which are factors associated with high serum AGE concentrations. The aim of this study was to evaluate the utility of a serum fluorescence AGE (F-AGE) method as a screening tool for gestational diabetes. Subjects and methods Serum samples from 225 GDM patients and 217 healthy pregnant women (healthy controls) were diluted 50-fold in phosphate-buffered saline, and the AGEs were estimated by fluorometric analysis (λEx 350 nm/ λEm 440 nm). Results No significant (P > 0.05) differences in AGE concentrations, expressed in Arbitrary Units (UA/mL × 104), were observed in the women with GDM or in the healthy controls. Furthermore, F-AGE concentrations did not change significantly during the pregnancy (12-32 weeks of gestation). Only the GDM group had a positive correlation (r = 0.421; P < 0.001) between F-AGEs and serum creatinine concentrations. Conclusion It was not possible to distinguish women with gestational diabetes from the healthy controls on the basis of serum F-AGE concentrations.

Type 2 diabetes-associated genetic variants of FTO, LEPR, PPARg, and TCF7L2 in gestational diabetes in a Brazilian population

ABSTRACT Objective Gestational diabetes mellitus (GDM) is a metabolic disorder that shares pathophysiologic features with type 2 diabetes mellitus. The aim of this study was to investigate the association of the polymorphisms fat mass and obesity-associated (FTO) rs1421085, leptin receptor (LEPR) rs1137100, rs1137101, peroxisome proliferator-activated receptor gamma (PPARg) rs1801282, and transcription factor 7-like 2 (TCF7L2) rs7901695 with GDM. Subjects and methods 252 unrelated Euro-Brazilian pregnant women were classified into two groups according to the 2015 criteria of the American and Brazilian Diabetes Association: healthy pregnant women (n = 125) and pregnant women with GDM (n = 127), matched by age. The polymorphisms were genotyped using fluorescent probes (TaqMan®). Results All groups were in Hardy-Weinberg equilibrium. The genotype and allele frequencies of the studied polymorphisms did not show significant differences between the groups (P > 0.05). In the healthy and GDM groups, the C allele frequencies (95% CI) of the FTO rs1421085 polymorphism were 36.8% [31-43%] and 35.0% [29-41%]; the G allele frequencies (95% CI) of the LEPR rs1137100 polymorphism were 24.8% [19-30%] and 22.8% [18-28%]; the G allele frequencies (95% CI) of the LEPR rs1137101 polymorphism were 43.6% [37-50%] and 42.9% [37-49%]; the G allele frequencies (95% CI) of the PPARg rs1801282 polymorphism were 7.6% [4-11%] and 8.3% [5-12%]; and the C allele frequencies (95% CI) of the TCF7L2 rs7901695 polymorphism were 33.6% [28-39%] and 39.0% [33-45%], respectively. Conclusion The studied polymorphisms were not associated with GDM in a Brazilian population.

The rs10885122 polymorphism of the adrenoceptor alpha 2A (ADRA2A) gene in Euro-Brazilians with type 2 diabetes mellitus

Objective To investigate the association of the rs10885122G>T polymorphism in the ADRA2A gene in a Euro-Brazilian sample of healthy (controls) and type 2 diabetic (T2D) subjects. Subjects and methods We used fluorescent probes (TaqMan) to genotype 241 subjects, that is, 121 healthy and 120 T2D subjects, who were classified based on the Brazilian Diabetes Association (2013) and American Diabetes Association (2014) criteria. Results The genotype and allele frequencies showed no significant (P > 0.05) difference between the two studied groups. The minor allele (T) frequencies (95%CI) for rs10885122 were 19% (14-24%) and 20% (15-26%) for healthy and T2D groups, respectively. Carriers of the T allele (genotypes GT+TT) were significantly associated (P = 0.016) with approximately a 7-kg body weight reduction compared with the genotype GG, which was only found in the T2D group. Conclusion The rs10885122G>T polymorphism of the ADRA2A gene was not associated with T2D in Euro-Brazilians, and carriers of the T allele had lower body weight in the presence of T2D. Arch Endocrinol Metab. 2015;59(1):29-33 .

RAGE receptor and its soluble isoforms in diabetes mellitus complications / O receptor RAGE e suas isoformas solúveis nas complicações do diabetes mellitus

Chronic hyperglycemia, which is present in all types of diabetes, increases the formation of advanced glycation end-products (AGEs). The interaction of AGEs with receptor of advanced glycation end-products (RAGE) initiates a cascade of pro-inflammatory and pro-coagulant processes that result in oxidative stress, stimulating the formation and accumulation of more AGE molecules. This cyclic process, denominated metabolic memory, may explain the persistency of diabetic vascular complications in patients with satisfactory glycemic control. The RAGE found in several cell membranes is also present in soluble isoforms (esRAGE and cRAGE), which are generated by alternative deoxyribonucleic acid splicing or by proteolytic cleavage. This review focuses on new research into these mediators as potential biomarkers for vascular complications in diabetes.

A hiperglicemia crônica, presente em todas as formas de diabetes, favorece a formação de produtos finais de glicação avançada (AGEs). A interação de AGEs com o receptor de produtos finais glicosilados (RAGE) inicia uma cascata de processos pró-inflamatórios e pró-coagulantes que resultam em estresse oxidativo, o qual estimula a formação e o acúmulo de maior quantidade de moléculas de AGEs. Esse processo cíclico, denominado memória metabólica, pode explicar por que, mesmo após um período de bom controle glicêmico, as complicações vasculares associadas ao diabetes não regridem. O RAGE fixado nas membranas de várias células também está presente em isoformas solúveis (esRAGE e cRAGE), geradas por processamento alternativo do ácido desoxirribonucleico (DNA) ou por clivagem proteolítica. Esta revisão aborda os novos estudos sobre a função desses mediadores como potenciais biomarcadores para as complicações vasculares no diabetes.

Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte â-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2). Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH) and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp) and modE2 (372 bp) genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.

Controle de qualidade da glicemia: um estudo interlaboratorial / Quality control for glucose determination: an interlaboratorial study

A performance de 36 laboratórios da regiäo Sul do Brasil, que voluntariamente participaram neste estudo, foi avaliada através da quantificaçäo da glicose. As amostras analisadas continham glicose, dissolvida em ácido benzóico saturado e 25 porcento de glicerol, nas concentraçöes de 20, 200 e 1000 mg/dL. O erro total máximo permitido nas determinaçöes foi estabelecido como 10 porcento do valor esperado ou ñ 6 mg/dL, o que for maior, como recomendado pelo critério do CLIA. As metodologias empregadas neste estudo foram glicose oxidase (80 porcento), hexoquinase (17 porcento) e glicose desidrogenase (3 porcento). Para a amostra de 200 mg/dL, 25,7 porcento dos laboratórios relataram resultados incorretos, sendo importante ressaltar que o aprimoramento da qualidade analítica em valores próximos a esta concentraçäo é necessário para o diagnóstico e monitoramento dos pacientes com diabetes. A amostra contendo a maior concentraçäo de glicose (1000 mg/dL) foi incorretamente reportada por 55,9 porcento dos participantes e os resultados mostraram uma forte tendência para resultados abaixo da concentraçäo real. Apenas 38,8 porcento dos laboratórios avaliados neste estudo atingiram simultaneamente resultados dentro dos limites de erro estabelecido nas três amostras analisadas