ABSTRACT
Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since their introduction in the neonatal screening of phenylketonuria almost 50 years ago. The range of its application has been extended to modern approaches, such as next-generation sequencing (NGS) for molecular genetic testing. This study aimed to evaluate the use of a standardized organic method for DNA extraction from DBS samples in the diagnostic setting.Methods: The clinical applicability of the method was tested using 3 samples collected from a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from 3 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and method performance was assessed by standard polymerase chain reaction, restriction length polymorphism, Sanger sequencing, and targeted NGS.Results: Results were compared with the ones obtained from DNA samples extracted following the internally validated in-house extraction protocol that used 6 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to having a lower cost per sample
Subject(s)
Humans , Infant, Newborn , Polymerase Chain Reaction/statistics & numerical data , Neonatal Screening , Sequence Analysis, DNA/statistics & numerical data , DNA/genetics , Dried Blood Spot Testing/statistics & numerical dataABSTRACT
Abstract Objective Most prenatal screening programs for toxoplasmosis use immunoassays in serum samples of pregnant women. Few studies assess the accuracy of screening tests in dried blood spots, which are of easy collection, storage, and transportation. The goals of the present study are to determine the performance and evaluate the agreement between an immunoassay of dried blood spots and a reference test in the serum of pregnant women from a population-based prenatal screening program for toxoplasmosis in Brazil. Methods A cross-sectional study was performed to compare the immunoassays Imunoscreen Toxoplasmose IgM and Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil)in dried blood spots with the enzymelinked fluorescent assay (ELFA, BioMérieux S.A., Lyon, France) reference standard in the serum of pregnant women from Minas Gerais Congenital Toxoplasmosis Control Program. Results The dried blood spot test was able to discriminate positive and negative results of pregnant women when comparedwith the reference test, with an accuracy of 98.2% for immunoglobulin G (IgG), and of 95.8% for immunoglobulin M (IgM). Conclusion Dried blood samples are easy to collect, store, and transport, and they have a good performance,making this a promisingmethod for prenatal toxoplasmosis screening programs in countries with continental dimensions, limited resources, and a high prevalence of toxoplasmosis, as is the case of Brazil.
Resumo Objetivo A maioria dos programas de triagem pré-natal para toxoplasmose utiliza imunoensaios em amostras de soro de gestantes. Poucos estudos avaliam a acurácia dos testes de triagem em amostras de sangue seco, que são de fácil coleta, armazenamento e transporte. Este estudo teve como objetivo determinar o desempenho e avaliar a concordância entre um imunoensaio em sangue seco e um teste de referência em soro de gestantes de um programa de rastreamento pré-natal de base populacional para toxoplasmose no Brasil. Métodos Realizou-se um estudo transversal para comparar os imunoensaios Imunoscreen Toxoplasmose IgM e Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil) em sangue seco com o padrão de referência ensaio fluorescente ligado a enzimas (enzyme-linked fluorescent assay, ELFA, BioMérieux S.A., Lion, França) no soro de gestantes do Programa de Controle de Toxoplasmose Congênita de Minas Gerais. Resultados O exame em sangue seco foi capaz de discriminar os resultados positivos e negativos das gestantes quando comparado ao teste de referência, com acurácia de 98,2% para imunoglobulina G (IgG), e de 95,8% para imunoglobulina M (IgM). Conclusão O sangue seco apresenta bom desempenho e é uma amostra de fácil coleta, armazenamento e transporte, o que o torna um método promissor para programas de triagem pré-natal de toxoplasmose em países com dimensões continentais, recursos limitados, e alta prevalência de toxoplasmose, como é o caso do Brasil.
Subject(s)
Humans , Female , Pregnancy , Toxoplasma/isolation & purification , Toxoplasmosis/diagnosis , Toxoplasmosis, Congenital/diagnosis , Immunoenzyme Techniques/methods , Dried Blood Spot Testing/methods , Prenatal Diagnosis , Toxoplasma/immunology , Brazil/epidemiology , Immunoglobulin G/blood , Immunoglobulin M/blood , Antibodies, Protozoan/blood , Toxoplasmosis/epidemiology , Toxoplasmosis, Congenital/epidemiology , Mass Screening , Population Surveillance , Prevalence , Cross-Sectional Studies , Pregnant WomenABSTRACT
A infecção pelo vírus da hepatite C (HCV) leva a cronicidade na maioria dos casos com possível evolução para cirrose e carcinoma hepatocelular. O diagnóstico laboratorial é feito através da pesquisa de anticorpos anti-HCV ou do HCV-RNA no soro ou plasma de indivíduos infectados. Porém o uso de amostras alternativas, tais como o sangue seco em papel filtro (SSPF) poderia facilitar o diagnóstico. A transmissão do vírus ocorre principalmente através do contato com sangue. A transmissão vertical ocorre em 4 a 8% dos casos, por este motivo é importante avaliar o impacto desta infecção e outras doenças infecciosas durante o pré-natal. No estado do Piauí, não é realizada a detecção de anti-HCV como teste de rotina no pré-natal em redes públicas. Logo, o objetivo deste trabalho é avaliar a prevalência da infecção pelo HCV e outras doenças infecciosas incluídas na triagem pré-natal em gestantes do Piauí. Entre outubro de 2018 e setembro de 2019, se realizou a busca de registros provenientes do sistema GAL (Gerenciador de Ambiente Laboratorial) referentes às amostras de SSPF que foram recebidas para a triagem pré-natal. Dados demográficos e resultados de diagnóstico para citomegalovírus, toxoplasmose, vírus da hepatite B, vírus da imunodeficiência humana e Treponema pallidum, foram coletados no sistema. Em relação ao HCV, as amostras de SSPF foram submetidas a detecção de antiHCV empregando ensaio imunoenzimático otimizado. As amostras reagentes foram submetidas a PCR convencional para detecção do HCV-RNA. No estudo foram incluídas 554 gestantes com média de 24,3± 6,7 anos. Entre as gestantes, 39,35% eram da zona urbana, 30,69% da cor parda e 21,12% estavam no 1º trimestre. Observamos uma prevalência de 1,08% de HCV, e nenhuma amostra possuía o RNA-HCV. As seguintes prevalências foram observadas: 89,94% de IgG anti-citomegalovírus e 0,2% de IgM anti-citomegalovírus; 63,71% para IgG anti-toxoplasmose; 0,39% para IgM anti-toxoplasmose; 0,39% de HBsAg, 0,18% de anti-HIV e 0,36% de anti-Treponema pallidum. Entre as gestantes do estudo, 486 realizaram duas visitas (t2), 62 realizaram três visitas (t3) e apenas 6 retornaram quatro vezes (t4). De acordo com as visitas, a prevalência de anti-HCV foi de 0,5%para uma visita, 0,2% para duas visitas, 0% para três e quatro consultas. Concluímos que a prevalência de HCV foi baixa nas gestantes do estudo, porém há necessidade de investir em programas de rastreio que podem auxiliar nas estratégias de redução de transmissão vertical.
Subject(s)
Hepatitis C , Perinatal Care , Coinfection , Dried Blood Spot TestingABSTRACT
Objective@#The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma.@*Method@#A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.@*Results@#Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log @*Conclusion@#The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.
Subject(s)
DNA, Viral/analysis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , HIV Infections/diagnosis , HIV-1/isolation & purification , Hepacivirus/isolation & purification , Hepatitis C/diagnosis , RNA, Viral/analysis , Sensitivity and Specificity , Specimen Handling/methods , Syphilis/diagnosis , Treponema pallidum/isolation & purificationABSTRACT
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to a deficiency of enzymes involved in cortisol biosynthesis. In more than 90% of cases, CAH is secondary to deleterious mutations in the CYP21A2 gene leading to 21-hydroxilase deficiency (21OHD). The CYP21A2 gene is located on the short arm of chromosome 6 (6p21·3) and encodes the cytochrome P450C21 enzyme. Neonatal screening programs detect the classic forms of CAH-21OHD quantifying 17OH-progesterone in dried blood spots (DBS). This test is very sensitive, but it has a low specificity, requiring a second sample to confirm the result. In these cases, a second-tier test in the same sample may be useful. Our aim was to evaluate a DNA extraction method from DBS and assess the performance of such DNA in the molecular analysis of the CYP21A2 gene mutations. Twelve individuals, who presumably had CAH based on the initial neonatal screening results, were analyzed using DNA extracted from freshly collected blood on EDTA and DBS. The CYP21A2 gene was analyzed by automated sequencing of all exons and intron boundaries and MLPA analysis in DBS. Molecular analysis results from both extraction methods were compared. In this study, we show that DNA extracted from neonatal screening DBS is a useful tool to define CYP21A2 gene mutations in 21-OHD diagnostic confirmation for the newborn screening program and that its results are comparable to traditional genotyping.
La hiperplasia suprarrenal congénita (HSC) es un desorden autosómico recesivo producido por la deficiencia de alguna de las enzimas involucradas en la biosíntesis de cortisol. Más del 90% se debe a mutaciones en el gen CYP21A2 que genera deficiencia de 21 hidroxilasa (21OHD). Este gen se encuentra en el brazo corto del cromosoma 6 (6p21·3) y codifica para la enzima citocromo P450C21. Los programas de pesquisa neonatal detectan la forma clásica de la HSC-21OHD cuantificando 17OH-progesterona en gota de sangre en papel de filtro (GSPF). Este test es muy sensible, pero tiene baja especificidad , por lo que se utiliza una segunda muestra para confirmar el resultado. En estos casos, una segunda determinación en la misma muestra podría ser de utilidad. Nuestro objetivo fue evaluar el método de extracción de ADN y posterior análisis molecular del gen CYP21A2 en muestras de GSPF. Analizamos doce individuos presumiblemente afectados por HSC en la pesquisa neonatal usando ADN extraído de sangre fresca recolectada sobre EDTA y de GSPF. Realizamos el análisis del gen CYP21A2 mediante secuenciación automática de todos los exones y regiones intrónicas flanqueantes y MLPA en GSPF, y comparamos los resultados con ambos métodos de extracción. En este estudio demostramos que el ADN extraído de GSPF es una herramienta muy útil para analizar las mutaciones del gen CYP21A2 en la confirmación diagnóstica de 21-OHD para los programas de pesquisa neonatal y que los resultados son comparables con la genotipificación tradicional.
Subject(s)
Humans , Male , Female , Infant, Newborn , Steroid 21-Hydroxylase/genetics , Neonatal Screening/methods , Adrenal Hyperplasia, Congenital/diagnosis , Adrenal Hyperplasia, Congenital/genetics , Dried Blood Spot Testing/methods , Mutation , Reference Values , Spectrophotometry , Polymerase Chain Reaction , Reproducibility of Results , Gestational Age , 17-alpha-Hydroxyprogesterone/analysis , AllelesABSTRACT
OBJECTIVE@#To explore effects of different delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots (DBS), so as to provide evidence for improving accurate and reliable detection by tandem mass spectrometry.@*METHODS@#A total of 1 254 616 newborn DBS samples in Newborn Screening Center of Zhejiang Province were delivered and stored at room temperature (group A, @*RESULTS@#The concentrations of amino acids and carnitines in the three groups were skewed, and the differences in amino acid and carnitine concentrations among groups were statistically significant (all @*CONCLUSIONS@#Cold-chain logistics system and storage in low temperature and low humidity can effectively reduce degradation of some amino acids and carnitines in DBS, improve the accuracy and reliability of detection, and thus ensures the quality of screening for neonatal metabolic diseases.
Subject(s)
Humans , Infant, Newborn , Amino Acids/analysis , Carnitine/analysis , Dried Blood Spot Testing/standards , Humidity , Neonatal Screening , Reproducibility of Results , Specimen Handling/standards , Tandem Mass Spectrometry , Temperature , Time FactorsABSTRACT
INTRODUCCIÓN: El diagnóstico de deficiencia de hormona de crecimiento (DHC) es difícil de establecer, y se puede asociar a serias complicaciones, especialmente en el período neonatal. La prueba de estímulo de secreción de hormona de crecimiento (HC) se considera de elección para el diagnóstico, pero presenta complicaciones metodológicas y se asocia a efectos adversos. Los neonatos presentan aumento de la secreción de HC de forma fisiológica, siendo una ventana diagnóstica. OBJETIVO: Evaluar si la muestra de sangre en papel filtro tomada en el período neonatal, en contexto del tamizaje neonatal de hipotiroidismo congénito y fenilcetonuria, permite diferenciar pacientes con DHC, de los que no la presentan. PACIENTES Y MÉTODO: Estudio de casos y controles mediante determinación de concentración de HC en sangre de papel filtro extraída en período neonatal, comparando controles con DHC con casos con deficiencia descartada. Se realizó extracción de la muestra del papel filtro, obteniendo dos discos de 0,125 pulgada por cada uno de los pacientes desde el centro de la mancha de sangre del papel, para un ELISA de HC humana altamente sensible basado en el uso de anticuerpos policlonales dirigidos contra la HC humana recombinante de 22kDa de peso molecular. RESULTADOS: Se obtuvo un total de 7 casos de DHC y 10 controles. La mediana de concentración de HC de papel filtro en los casos es 2,0 ng/ml (Rango intercuartil 3,6 ng/ml) y controles 2,05 ng/mL (RIC 2,0 ng/ml), U de Mann-Withney 30,5 (p = 0,68). Los dos casos con deficiencia de hormonas hipofisarias múltiples (DHHM) presentan concentraciones menores a 1 ng/ml. CONCLUSIÓN: La muestra de papel filtro no permitió diferenciar a los pacientes con DHC de los casos controles, aunque los casos con DHHM presentaron concentraciones mucho menores, en comparación a la deficiencia de hormona de crecimiento aislada (DHCA).
INTRODUCTION: The diagnosis of growth hormone deficiency (GHD) is difficult to determine, and could be associated with severe complications, especially in the neonatal period. The stimulation test of growth hormone (GH) secretion is considered the gold standard for diagnosis, but it has methodological complications and is associated with adverse effects. Neonates present physiological increased secretion of GH, representing a diagnostic window. OBJECTIVE: To evaluate if the dried blood spot on filter paper obtained in the neonatal period, as part of a neonatal screening for con genital hypothyroidism and phenylketonuria, allows differentiating patients with GHD from those who do not have it. PATIENTS AND METHOD: Study of cases and controls by measuring the GH concen tration in dried blood spot on filter paper obtained in the neonatal period, comparing controls with GHD with cases with discarded deficiency. The sample was extracted from the filter paper, obtaining two 0.125 inch discs per each patient from the center of the blood spot on the paper, for a highly sen sitive ELISA assay for human GH based on the use of polyclonal antibodies against 22 kDa recom binant human GH. RESULTS: Seven cases of GHD and ten controls were obtained. The median GH concentration of the dried blood spot in the cases is 2.0 ng/ml (Interquartile range 3.6 ng/ml) and 2.05 ng/ml (Interquartile range 2.0 ng/ml) in the controls, Mann-Whitney U test 30.5 (p = 0.68). The two cases with multiple pituitary-hormone deficiency (MPHD) present concentrations lower than 1 ng/ml. CONCLUSION: The dried blood spot sample did not differentiate GHD patients from control cases, although MPHD cases present much lower concentrations compared to isolated growth hor mone deficiency (IGHD).
Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Child , Neonatal Screening , Human Growth Hormone/deficiency , Dried Blood Spot Testing , Growth Disorders/diagnosis , Hypopituitarism/diagnosis , Biomarkers/blood , Case-Control Studies , Human Growth Hormone/blood , Dwarfism, Pituitary/diagnosis , Dwarfism, Pituitary/blood , Growth Disorders/etiology , Growth Disorders/blood , Hypopituitarism/complications , Hypopituitarism/bloodABSTRACT
Abstract Introduction: Anti-nucleosome and anti-C1q antibodies demonstrated an association with the development of glomerulonephritis in systemic lupus erythematosus (SLE). Some investigators have proposed that monitoring anti- C1q and anti-nucleosome antibodies might be valuable for making predictions about lupus nephritis (LN) and assessment of disease activity as a non-invasive biological marker of renal disease. Objectives: The current study was proposed to investigate the presence of anti-C1q and anti-nucleosome antibodies in the sera of Egyptian patients with SLE and their association with LN. Methods: Eighty patients with SLE were included. Patients were classified into, a LN group including 40 cases with active LN (based on the results of renal biopsy and renal SLEDAI≥4) and a non renal SLE group including 40 patients (with no clinical or laboratory evidence of renal involvement that were attributed in the past or present to SLE). They were subjected to full medical history taking, clinical examination, routine laboratory investigations, measurement of antinuclear antibody (ANA), anti-ds DNA, anti-C1q & anti-nucleosome antibodies. Results: Anti-C1q antibody showed a statistically significant association with the presence of vasculitis and nephritis while anti-nucleosome antibody didn't show a significant association with the presence of any clinical features. Double positivity of anti-nucleosome and anti-C1q antibodies showed a statistically significant association with the presence of vasculitis and photosensitivity, high ECLAM score, elevated ESR, low serum albumin and low C3 levels. Conclusion: Serum anti-C1q antibody has a significant association with LN while double positive antibodies have a significant association with vasculitis and low C3 levels in Egyptian patients with SLE.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Pulmonary Medicine/methods , Glycogen Storage Disease Type II/complications , Glycogen Storage Disease Type II/diagnosis , Dried Blood Spot Testing/standards , Late Onset Disorders/diagnosis , Lung Diseases/complications , Biopsy , Glycogen Storage Disease Type II/blood , Glycogen Storage Disease Type II/enzymology , Early Diagnosis , alpha-Glucosidases/metabolism , Late Onset Disorders/blood , Late Onset Disorders/enzymology , Italy , Lung Diseases/blood , Muscles/surgery , Muscles/enzymologyABSTRACT
OBJECTIVE@#To assess the value of dry blood spot tandem mass spectrometry for the diagnosis of autism spectrum disorder (ASD).@*METHODS@#Peripheral blood samples of 277 autistic children were collected. Their amino acid and carnitine profiles were detected by liquid chromatography tandem mass spectrometry. Urine samples of suspected patients were collected for verification by gas chromatography mass spectrometry. Blood samples were also taken for genetic testing.@*RESULTS@#Of the 277 children with ASD, 19 (6.9%) were suspected to be with inborn error of metabolism (IEM), which included 6 cases with amino acidemia, 9 with organic acidemia and 4 with fatty acidemia. Three cases of phenylketonuria, one case of homocysteinemia, one case of propionemia, one case of methylmalonic acidemia, one case of glutaric acidemia, one case of isovaleric acidemia, one case of argininemia, one case of citrullinemia I and four cases of primary carnitine deficiency were confirmed by genetic testing, which yielded an overall diagnostic rate of 5.1% (14/277).@*CONCLUSION@#Our result has provided further evidence for the co-occurrence of ASD and IEM. Tandem mass spectrometry has a great value for the diagnosis and treatment of ASD in childhood.
Subject(s)
Child , Humans , Amino Acid Metabolism, Inborn Errors , Diagnosis , Autism Spectrum Disorder , Diagnosis , Dried Blood Spot Testing , Gas Chromatography-Mass Spectrometry , Metabolism, Inborn Errors , Diagnosis , Tandem Mass SpectrometryABSTRACT
ABSTRACT Vaccination against the hepatitis A virus (HAV) administered in two doses has been used effectively in universal child immunization programs in several countries. A single-dose vaccination was adopted in some low-income countries in an attempt to reduce costs without losing effectiveness. In 2014, single-dose universal vaccination was introduced in Brazil for children aged two years. Since such strategy is still not universally accepted, its efficacy should be compared to the two-dose strategy. To assess the humoral response after the single-dose HAV vaccination schedule, a cross-sectional study was conducted in Primavera do Leste, in Mato Grosso state, Central Brazil, including 265 children vaccinated through the National Immunization Program. Blood was collected by using a digital puncture and further applied to filter paper cards. Anti-HAV was detected in 218 out of 265 dried blood spots (DBS). Blood venous samples were collected from 34 out of 47 children who were not anti-HAV positive in DBS samples. Eighteen of them tested positive for anti-HAV, giving a final score of 93.6% (236/252) of seropositivity. In conclusion, this study demonstrated a high rate of anti-HAV positivity in the short term after single-dose hepatitis A vaccination in the population investigated. Moreover, the DBS was shown to be a reliable tool for detecting anti-HAV antibodies.
Subject(s)
Humans , Male , Female , Child , Mass Vaccination/methods , Hepatitis A Vaccines/administration & dosage , Hepatitis A Antibodies/blood , Hepatitis A/prevention & control , Brazil/epidemiology , Program Evaluation , Logistic Models , Seroepidemiologic Studies , Retrospective Studies , Immunoenzyme Techniques , Immunization Schedule , Hepatitis A Virus, Human/immunology , Hepatitis A Vaccines/immunology , Dried Blood Spot Testing , Hepatitis A/epidemiologyABSTRACT
Resumo: Introdução: A classe de drogas de abuso conhecida como novas substâncias psicoativas ou, ainda, drogas desenhadas (do inglês new psychoactive substances (NPS) ou designer drugs) é composta por substâncias químicas obtidas a partir de alterações estruturais de substâncias ou mistura de substâncias psicoativas já existentes (e ilícitas), a fim de mimetizar e/ou potencializar os efeitos proporcionados por estas, com a vantagem de circundar a legislação antidrogas vigente. Dentro da classe das NPS, os grupos de maior destaque atualmente são das catinonas sintéticas, dos canabinóides sintéticos e dos NBOMes (feniletilaminas N-2-metoxibenzil substituídas). As catinonas sintéticas, vendidas pela internet como "sais de banho" ou bath salts, são substâncias estruturalmente relacionadas a catinona, um alcalóide presente no Catha edulis (Khat), com propriedades estimulantes. A classe dos NBOMes é derivada da classe dos alucinógenos 2C, a partir da adição de um grupo N-2-metoxibenzil na amina primária. Os NBOMes possuem ação agonista nos receptores de serotonina, especialmente do subtipo 5-HT2A, o que confere os efeitos alucinógenos. Objetivos: Desenvolver e validar métodos para identificação e quantificação de algumas das NPS de maior relevância em amostras de manchas de sangue seco em papel (do inglês dried blood spots, DBS) e comparar a estabilidade entre dois tipos de amostras (DBS e sangue total), em três diferentes temperaturas (ambiente, 4 °C e -20 °C) e períodos de armazenamento. Metodologia: As amostras de DBS e sangue total foram analisadas em cromatógrafo líquido acoplado à espectrômetro de massas com analisador de massas triplo quadrupolar (LC-MS/MS), utilizando os métodos primeiramente desenvolvidos e validados para cada classe de NPS (catinonas sintéticas e NBOMes). Resultados: A técnica de DBS apresentou um aumento significativo da estabilidade das NPS em comparação à técnica convencional de armazenamento de sangue total. Para os NBOMes, observou-se que os compostos eram estáveis no DBS por período de tempo de até 6 meses, nas três temperaturas estudadas, enquanto que no sangue total, os analitos tiveram uma queda maior que 20% da concentração em 15 ou 30 dias em temperatura ambiente e 180 dias para 4 °C. Para as catinonas, em temperatura ambiente, observou-se baixa estabilidade nas duas matrizes. Porém, para armazenamento à 4 °C, observou-se uma queda na concentração inicial maior que 20% com 60 e 90 dias para mefedrona e benzedrona em DBS, respetivamente, contra 45 e 25 dias. Catinonas que possuem grupamento metilenodioxi em sua estrutura, como a butilona e a pentilona, foram mais estáveis, independente da matriz, em comparação àquelas com grupamento alquila no anel aromático, como mefedrona e benzedrona. Conclusão: A técnica de DBS se mostrou vantajosa para análises forenses em comparação à técnica convencional de armazenamento de sangue total. Além de facilitar o armazenamento (por ocupar menos espaço), sua extração se torna mais rápida e facilitada, já que envolve menos etapas, além de tornar possível a identificação dos analitos de interesse por um maior período de tempo, podendo facilmente ser aplicada na rotina laboratorial(AU)
Abstract: Introduction: New psychoactive substances (NPS) or designer drugs is a class of drugs of abuse composed of chemical substances obtained from structural alterations of substances or mixture of existing psychoactive substances (and illicit), in order to mimic and/or maximize their effects, with the advantage of circumventing existing anti-drug legislation. Within the NPS class, the most prominent groups currently are synthetic cathinones, synthetic cannabinoids and NBOMes (phenylethylamines N-2-methoxybenzyl substituted). Synthetic cathinones, sold by the internet as "bath salts", are substances structurally related to cathinone, an alkaloid present in Catha edulis (Khat), with stimulant properties. The class of NBOMes is derived from the hallucinogen class 2C, from the addition of an N-2-methoxybenzyl group to the primary amine. NBOMes have agonist action at serotonin receptors, especially the 5-HT2A subtype, which confers the hallucinogenic effects. Objectives: To develop and validate methods to identify and quantify some of the most relevant NPS in dried blood spots (DBS) samples and to compare the stability between two matrixes (DBS and whole blood), at three different temperatures (ambient, 4 °C and -20 °C) and storage periods. Method: DBS and whole blood samples were analyzed using a liquid chromatography tandem mass spectrometer with a triple quadrupole analyzer (LC-MS/MS), using the developed and validated methods for each class of NPS (synthetic cathinones and NBOMes). Results: The DBS technique showed a significant increase in the NPS stability compared to the conventional whole blood storage technique. For the NBOMes, the compounds were found to be stable in DBS for a time period of up to 6 months, at the three temperatures studied, whereas in whole blood, the analytes had a decreased higher than 20% of the initial concentration in 15 or 30 days at room temperature and 180 days at 4 °C. For the cathinones, at room temperature, low stability was observed in both matrixes. However, for storage at 4 °C, the initial concentration decreased more than 20% at 60 and 90 days for mephedrone and benzedrone in DBS, respectively, against 45 and 25 days. Cathinones having methylenedioxy group in their structure, such as butylone and pentylone, were more stable, independent of the matrix, compared to those with alkyl group on the aromatic ring, such as mephedrone and benzedrone. Conclusion: The DBS technique proved to be advantageous for forensic analysis compared to the conventional storage technique. In addition to occupying less storage space, DBS extraction becomes faster and easier, since it involves fewer steps, besides to make possible to identify the analytes of interest for a longer period of time, which can easily be applied in the laboratory routine(AU)
Subject(s)
Humans , Designer Drugs , Dried Blood Spot Testing , Forensic Toxicology , Chromatography, Liquid , Mass Spectrometry , Phenethylamines , Tandem Mass SpectrometryABSTRACT
Introducción: La enfermedad de Gaucher es un trastorno metabólico por deficiencia o ausencia de enzima ß-Glucosidasa Ácida. El diagnóstico se sospecha clínicamente, pero requiere confirmación mediante medición, en leucocitos (estándar de oro) o en sangre seca sobre papel de filtro, de la actividad de la enzima ß-Glucosidasa Ácida. Objetivo: Evaluar la validez de la medición de la actividad de la enzima ß-Glucosidasa Ácida en sangre seca en papel de filtro, comparada con el estándar de oro, para el diagnóstico de enfermedad de Gaucher en pacientes con sospecha clínica. Metodología: Se hizo una revisión sistemática de literatura. Se construyó y validó una pregunta PICO. Se usó una estrategia de búsqueda genérica con base en los términos clave (Gaucher Disease y Dried Blood Spot Analysis). Dos evaluadores independientes revisaron, evaluaron la calidad y extrajeron la información de los artículos. Resultados: Descartando los duplicados, se obtuvieron 47 artículos. Se evaluaron los textos completos de cuatro, y tres de ellos fueron excluidos al aplicar criterios de inclusión y exclusión. El artículo incluido tuvo una calidad excelente y mostró que la actividad enzimática de la glucocerebrosidasa en sangre seca tuvo sensibilidad de 82.3% y especificidad de 94.0% con un punto de corte de 0.0-2.75 y sensibilidad de 88.2% y especificidad de 88.5% con un punto de corte de 0.0-4.4. Conclusiones: La medición enzimática de la ß-Glucosidasa Ácida en sangre seca es una excelente prueba para diagnóstico inicial de enfermedad de Gaucher. Sin embargo, no es una prueba concluyente. [Vera-Cala LM, Serrano-Gómez SE, Córtes A, Estrada I, Gáfaro A. Validez de la prueba de actividad enzimática de la glucocerebrosidasa para el diagnóstico de enfermedad de Gaucher, revisión sistemática. MedUNAB 2017; 20(2): 201-206].
Introduction: The Gaucher disease is a metabolic disorder due to deficiency or absence of acid ß-glucosidase enzyme. The diagnosis is clinically suspected, but requires confirmation by measuring the activity of acid ß-glucosidase enzyme in leukocytes (gold standard) or in dried blood on filter paper. Objective: To assess the validity of the measurement of acid ß-glucosidase enzyme activity in dried blood on filter paper compared to the standard of gold, for the diagnosis of Gaucher disease in patients with clinical suspicion. Methodology: A systematic review of literature was carried out. A PICO question was constructed and validated. A generic search strategy was used based on the key terms (Gaucher Disease and Dried Blood Spot Analysis). Two independent evaluators reviewed and assessed the quality, and extracted information from the articles. Results: Discarding the duplicates, 47 articles were obtained. Four complete texts were evaluated, and three of them were excluded when applying inclusion and exclusion criteria. The article that was included had an excellent quality and showed that the enzymatic activity of glucocerebrosidase in dried blood had a sensitivity of 82.3%, a specificity of 94.0% with a cut-off of 0.0-2.75, a sensitivity of 88.2% and specificity of 88.5% with a cut-off point of 0.0-4.4. Conclusions: The enzymatic measurement of acid ß-glucosidase in dried blood is an excellent test for an initial diagnosis of Gaucher disease; however, it is not a conclusive proof. [Vera-Cala LM, Serrano-Gómez SE, Córtes A, Estrada I,Gáfaro A. Validity of the Enzymatic Activity Test of Glucocerebrosidase for the Diagnosis of Gaucher Disease, a Systematic Review. MedUNAB 2017; 20(2): 201-206].
Introdução: A doença de Gaucher é um transtorno metabólico devido a deficiência ou ausência de enzima de ß-Glucosidase. O diagnóstico é clinicamente suspeitado, mas requer confirmação medindo, em leucócitos (padrão-ouro) ou em sangue seco em papel de filtro, a atividade de enzima de ß-Glucosidase. Objetivo: Avaliar a validade da medida da atividade de enzima de ß-Glucosidase em sangue seco em papel de filtro, em comparação com o padrão-ouro, para o diagnóstico de doença de Gaucher em pacientes com suspeita clínica. Metodologia: Foi feita uma revisão sistemática da literatura. Uma questão PICO foi construída e validada. Uma estratégia de pesquisa genérica foi utilizada com base nos títulos-chave (Doença de Gaucher e Análise de Pontos de Sangue Seco). Avaliaram dois avaliadores independentes, avaliaram a qualidade e extraíram informações dos artigos. Resultados: Descartando as duplicatas, foram obtidos 47 artigos. Os textos completos de quatro foram avaliados e três deles foram excluídos ao aplicar critérios de inclusão e exclusão. O artigo incluído teve uma excelente qualidade e mostrou que a atividade enzimática da glucocerebrosidase em sangue seco teve sensibilidade de 82.3% e especificidade de 94.0% com um corte de 0.0-2.75 e sensibilidade de 88. 2% e especificidade de 88.5% com ponto de corte de 0.0-4.4. Conclusões: A medida enzimática da ß-glucosidase ácida em sangue seco é um excelente teste para o diagnóstico inicial de doença de Gaucher. No entanto, não é uma prova conclusiva. [Vera-Cala LM, Serrano-Gómez SE, Córtes A, Estrada I, Gáfaro A. Validade do teste de atividade enzimática da glucocerebrosidase para o diagnóstico da doença de Gaucher, revisão sistemática. MedUNAB 2017; 20(2): 201-206].
Subject(s)
Gaucher Disease , Sensitivity and Specificity , Diagnosis , Dried Blood Spot Testing , Glucosylceramidase , LeukocytesABSTRACT
Abstract Objective To apply, in Brazil, the T-cell receptor excision circles (TRECs) quantification technique using real-time polymerase chain reaction in newborn screening for severe combined immunodeficiency and assess the feasibility of implementing it on a large scale in Brazil. Methods 8715 newborn blood samples were collected on filter paper and, after DNA elution, TRECs were quantified by real-time polymerase chain reaction. The cutoff value to determine whether a sample was abnormal was determined by ROC curve analysis, using SSPS. Results The concentration of TRECs in 8,682 samples ranged from 2 to 2,181 TRECs/µL of blood, with mean and median of 324 and 259 TRECs/µL, respectively. Forty-nine (0.56%) samples were below the cutoff (30 TRECs/µL) and were reanalyzed. Four (0.05%) samples had abnormal results (between 16 and 29 TRECs/µL). Samples from patients previously identified as having severe combined immunodeficiency or DiGeorge syndrome were used to validate the assay and all of them showed TRECs below the cutoff. Preterm infants had lower levels of TRECs than full-term neonates. The ROC curve showed a cutoff of 26 TRECs/µL, with 100% sensitivity for detecting severe combined immunodeficiency. Using this value, retest and referral rates were 0.43% (37 samples) and 0.03% (3 samples), respectively. Conclusion The technique is reliable and can be applied on a large scale after the training of technical teams throughout Brazil.
Resumo Objetivo Aplicar no Brasil a técnica de quantificação de T-cell Receptor Excision Circles (TRECs) por PCR em tempo real para triagem neonatal de imunodeficiência combinada grave (SCID) e avaliar se é possível fazê-la em grande escala em nosso país. Métodos Foram coletadas em papel filtro 8.715 amostras de sangue de recém-nascidos e, após eluição do DNA, os TRECs foram quantificados por PCR em tempo real. O valor de corte para determinar se uma amostra é anormal foi determinado pela análise de curva ROC com o programa SSPS. Resultados A concentração de TRECs em 8.682 amostras analisadas variou entre 2 e 2.181 TRECs/µL de sangue, com média e mediana de 324 e 259 TRECs/µL, respectivamente. Das amostras, 49 (0,56%) ficaram abaixo do valor de corte (30 TRECs/µL) e foram requantificadas. Quatro (0,05%) mantiveram resultados anormais (entre 16 e 29 TRECs/µL). Amostras de pacientes com diagnóstico clínico prévio de SCID e síndrome de DiGeorge foram usadas para validar o ensaio e todas apresentaram concentração de TRECs abaixo do valor de corte. Recém-nascidos prematuros apresentaram menores níveis de TRECs comparados com os nascidos a termo. Com o uso da curva ROC em nossos dados, chegamos ao valor de corte de 26 TRECs/µL, com sensibilidade de 100% para detecção de SCID. Com o uso desse valor, as taxas de repetição e encaminhamento ficaram em 0,43% (37 amostras) e 0,03% (3 amostras), respectivamente. Conclusão A técnica é factível e pode ser implantada em grande escala, após treinamento técnico das equipes envolvidas.
Subject(s)
Humans , Male , Female , Infant, Newborn , Receptors, Antigen, T-Cell/blood , Neonatal Screening/methods , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/blood , Reference Values , Time Factors , Brazil , Receptors, Antigen, T-Cell/genetics , Reproducibility of Results , Sensitivity and Specificity , Age Factors , Statistics, Nonparametric , Dried Blood Spot Testing , Real-Time Polymerase Chain ReactionABSTRACT
Resumo Introdução: A identificação precoce da doença renal crônica (DRC) por meio de amostras de sangue e urina é preconizada em populações de risco devido à elevada morbimortalidade. Objetivo: Apresentamos um teste simples e inovador para dosar a creatinina coletada em gota de sangue seca em papel filtro (PF). Métodos: Cento e seis pessoas em risco de DRC foram rastreadas com avaliação de dados clínicos, exame físico e coleta de sangue de forma convencional e em PF. Com os dados obtidos, foi estimada a taxa de filtração glomerular (e-TFG). Foi considerado diagnóstico de DRC a e-TFG < 60 ml/min. Resultados: A idade dos participantes foi de 57 ± 12 anos, 78 (73,5%) eram mulheres, 43 brancos (40,5%), 36 pardos (34%) e 27 negros (25,5%). O índice de massa corpórea foi de 29,5 ± 6,9 kg/m2, a pressão arterial sistólica foi de 125 mmHg (120-140 mmHg) e a pressão arterial diastólica de 80 mmHg (70-80 mmHg). A sensibilidade pela equação CKD-EPI foi de 94%, a especificidade 55%, o valor preditivo positivo foi de 94%, o valor preditivo negativo de 55% e a acurácia de 90%. A estatística de Bland-Altman mostrou que as diferenças entre os valores de creatinina dos dois testes estão numa faixa relativamente estreita (+ 0,68 mg/dL e -0,55mg/dL) para um desvio padrão de ± 1,96 mg/dL. Conclusão: A dosagem da creatinina coletada em gota de sangue em PF é um teste diagnóstico simples de ser realizado, pouco invasivo e que apresentou uma ótima acurácia, podendo ser útil para rastrear DRC.
Abstract Introduction: Chronic kidney disease (CKD) screening is advisable due to its high morbidity and mortality and is usually performed by sampling blood and urine. Objective: Here we present an innovative and simpler method, by measuring creatinine on a dry blood spot on filter paper. Methods: One-hundred and six individuals at high risk for CKD were enrolled. The creatinine values obtained using both tests and the demographic data of each participant allowed us to determinate the eGFR. The adopted cutoff for CKD was an eGFR < 60 ml/min. Results: Mean age was 57 ± 12 years, 74% were female, 40% white, and 60% non-white. Seventy-six percent were hypertensive, 30% diabetic, 37% had family history of CKD, and 22% of smoking. The BMI was 29.5 ± 6.9 kg/m2, median systolic blood pressure was 125 mmHg (IQR 120-140 mmHg) and median diastolic blood pressure was 80 mmHg (IQR 70-80 mmHg). According to MDRD equation, sensitivity was 96%, specificity 55%, predictive positive value 96%, predictive negative value 55% and accuracy 92%. By the CKD-EPI equation the sensitivity was 94%, specificity 55%, predictive positive value 94%, predictive negative value 55% and accuracy 90%. A Bland and Altman analysis showed a relatively narrow range of creatinine values differences (+ 0.68mg/dl to -0.55mg/dl) inside the ± 1.96 SD, without systematic differences. Conclusion: Measurement of creatinine on dry blood sample is an easily feasible non-invasive diagnostic test with good accuracy that may be useful to screen chronic kidney disease.
Subject(s)
Humans , Male , Female , Middle Aged , Aged , Creatinine/blood , Renal Insufficiency, Chronic/diagnosis , Dried Blood Spot Testing , Kidney Function TestsABSTRACT
Background: There is a paucity of data on the prevalence of hepatitis C virus (HCV) in children, particularly in sub-Saharan Africa. A major obstacle in resource-limited settings for polymerase chain reaction (PCR) testing is the necessity for specimen transportation and storage at low temperatures. There are numerous recent studies of using real-time HCV PCR for diagnosis and screening of plasma and serum, but few have looked at using dried blood spot (DBS)specimens. Objectives: The aim of this study was to optimise a real-time HCV PCR method to detect HCV RNA from infant DBS specimens for use as a tool for HCV surveillance in KwaZulu-Natal, South Africa. Method: The LightCycler® 2.0 instrument was used for the HCV PCR using the LightCycler® RNA Master SYBR Green I kit. Template volume, primer concentration and primer annealing temperatures were optimised and the method was used on 179 DBS specimens from HIV-exposed infants in KwaZulu-Natal. Results: Primer concentrations adjusted to 0.25 µM and a template volume of 10 µL improved the PCR amplification. Primer annealing temperatures lowered from 65°C to 58°C resulted in higher quantities of amplified PCR product. The limit of detection of the optimised HCV PCR assay was between 1200 IU/mL and 3580 IU/mL of HCV RNA. HCV was not detected in any of the 179 DBS specimens.Conclusion: The optimised real-time HCV PCR on infant DBS specimens performed well, but HCV was not found in this surveillance study. HIV infection may have little impact on the vertical transmission of HCV in this region
Subject(s)
Dried Blood Spot Testing , HIV Infections , Infant , South AfricaABSTRACT
Background: Poor quality dried blood spot (DBS) specimens are usually rejected by virology laboratories; affecting early infant diagnosis of HIV. The practice of combining two incompletely-filled DBS in one specimen preparation tube during pre-analytical specimen processing (i.e.; the two-spot method) has been implemented to reduce the number of specimens being rejected for insufficient volume.Objectives: This study analysed laboratory data to describe the quality of DBS specimens and the use of the two-spot method over a one-year period; then validated the two-spot method against the standard (one-spot) method.Methods: Data on HIV-1 PCR test requests submitted in 2014 to the Department of Virology at Inkosi Albert Luthuli Central Hospital in KwaZulu-Natal province; South Africa were analysed to describe reasons for specimen rejection; as well as results of the two-spot method. The accuracy; lower limit of detection and precision of the two-spot method were assessed.Results: Of the 88 481 specimens received; 3.7% were rejected for pre-analytical problems. Of those; 48.9% were rejected as a result of insufficient specimen volume. Two health facilities had significantly more specimen rejections than other facilities. The two-spot method prevented 10 504 specimen rejections. The Pearson correlation coefficient comparing the standard to the two-spot method was 0.997.Conclusions: The two-spot method was comparable with the standard method of pre-analytical specimen processing. Two health facilities were identified for targeted retraining on specimen quality. The two-spot method of DBS specimen processing can be used as an adjunct to retraining; to reduce the number of specimens rejected and improve linkage to care
Subject(s)
HIV-1 , Dried Blood Spot Testing , Polymerase Chain Reaction , South Africa , Specimen HandlingABSTRACT
BACKGROUND: A newborn screening (NBS) program has been utilized to detect asymptomatic newborns with inherited metabolic diseases (IMDs). There have been some bottlenecks such as false-positives and imprecision in the current NBS tests. To overcome these issues, we developed a multigene panel for IMD testing and investigated the utility of our integrated screening model in a routine NBS environment. We also evaluated the genetic epidemiologic characteristics of IMDs in a Korean population. METHODS: In total, 269 dried blood spots with positive results from current NBS tests were collected from 120,700 consecutive newborns. We screened 97 genes related to NBS in Korea and detected IMDs, using an integrated screening model based on biochemical tests and next-generation sequencing (NGS) called NewbornSeq. Haplotype analysis was conducted to detect founder effects. RESULTS: The overall positive rate of IMDs was 20%. We identified 10 additional newborns with preventable IMDs that would not have been detected prior to the implementation of our NGS-based platform NewbornSeq. The incidence of IMDs was approximately 1 in 2,235 births. Haplotype analysis demonstrated founder effects in p.Y138X in DUOXA2, p.R885Q in DUOX2, p.Y439C in PCCB, p.R285Pfs*2 in SLC25A13, and p.R224Q in GALT. CONCLUSIONS: Through a population-based study in the NBS environment, we highlight the screening and epidemiological implications of NGS. The integrated screening model will effectively contribute to public health by enabling faster and more accurate IMD detection through NBS. This study suggested founder mutations as an explanation for recurrent IMD-causing mutations in the Korean population.
Subject(s)
Humans , Infant, Newborn , Computational Biology , DNA/chemistry , Dried Blood Spot Testing , Galactokinase , Genomics , Haplotypes , High-Throughput Nucleotide Sequencing , Incidence , Membrane Proteins/genetics , Metabolic Diseases/diagnosis , Metabolism, Inborn Errors/diagnosis , Mitochondrial Membrane Transport Proteins/genetics , Neonatal Screening , Polymorphism, Genetic , Republic of Korea/epidemiology , Sequence Analysis, DNAABSTRACT
As dried blood spots (DBSs) have various advantages over conventional venous blood sampling, some assays for detection of one or two anti-tuberculosis (TB) drugs in DBSs have been developed. However, there are no assays currently available for the simultaneous measurement of three or more anti-TB drugs in DBSs. In this study, we developed and evaluated a multiplex method for detecting nine anti-TB drugs including streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide, and linezolid in DBSs by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Seventy-nine patient samples of DBS were analyzed on the UPLC-MS/MS system. All drug concentrations were determined within 4 min, and assay performance was evaluated. All drugs were clearly separated without ion suppression. Within-run and between-run precisions were 1.7-13.0% and 5.7-17.0%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.06-0.6 and 0.5-5.0 µg/mL, respectively. Linearity was acceptable at five level concentrations for each drug. Correlations between drug concentrations in plasma and DBSs by using Passing-Bablock regression and Pearson's rho (ρ, 0.798-0.989) were acceptable. In conclusion, we developed a multiplex assay to measure nine second-line anti-TB drugs in DBSs successfully. This assay provided convenient and rapid drug quantification and could have applications in drug monitoring during treatment.
Subject(s)
Humans , Antitubercular Agents/blood , Chromatography, High Pressure Liquid , Dried Blood Spot Testing , Limit of Detection , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
AbstractBackground:Fabry disease is a lysosomal storage disease caused by enzyme α-galactosidase A deficiency as a result of mutations in the GLA gene. Cardiac involvement is characterized by progressive left ventricular hypertrophy.Objective:To estimate the prevalence of Fabry disease in a population with left ventricular hypertrophy.Methods:The patients were assessed for the presence of left ventricular hypertrophy defined as a left ventricular mass index ≥ 96 g/m2 for women or ≥ 116 g/m2 for men. Severe aortic stenosis and arterial hypertension with mild left ventricular hypertrophy were exclusion criteria. All patients included were assessed for enzyme α-galactosidase A activity using dry spot testing. Genetic study was performed whenever the enzyme activity was decreased.Results:A total of 47 patients with a mean left ventricular mass index of 141.1 g/m2 (± 28.5; 99.2 to 228.5 g/m2] were included. Most of the patients were females (51.1%). Nine (19.1%) showed decreased α-galactosidase A activity, but only one positive genetic test − [GLA] c.785G>T; p.W262L (exon 5), a mutation not previously described in the literature. This clinical investigation was able to establish the association between the mutation and the clinical presentation.Conclusion:In a population of patients with left ventricular hypertrophy, we documented a Fabry disease prevalence of 2.1%. This novel case was defined in the sequence of a mutation of unknown meaning in the GLA gene with further pathogenicity study. Thus, this study permitted the definition of a novel causal mutation for Fabry disease - [GLA] c.785G>T; p.W262L (exon 5).
ResumoFundamento:A doença de Fabry é uma doença lisossomal de sobrecarga provocada pela deficiência da enzima α-galactosidase A como resultado de mutações no gene GLA. O envolvimento cardíaco carateriza-se por hipertrofia ventricular esquerda progressiva.Objetivo:Estimar a prevalência da doença de Fabry numa população com hipertrofia ventricular esquerda.Métodos:Os doentes foram avaliados para a presença de hipertrofia ventricular esquerda definida por massa do ventrículo esquerdo indexada como ≥ 96 g/m2 para mulheres ou ≥ 116 g/m2 para homens. Estenose aórtica severa e hipertensão arterial, com hipertrofia ventricular esquerda discreta, foram critério de exclusão. Todos os doentes incluídos foram avaliados para a atividade da enzima α-galactosidase A com testes de gota seca. No caso de atividade enzimática diminuída, realizava-se estudo genético.Resultados:Foram incluídos 47 doentes com uma média de massa indexada de 141,1 g/m2 (± 28,5; 99,2 a 228,5 g/m2]. A maioria (51,1%) dos doentes era do sexo feminino. Nove deles (19,1%) tinham diminuição da atividade da α-galactosidase A, mas apenas um teste genético foi positivo − [GLA] c.785G>T; p.W262L (éxon 5), uma mutação não descrita na literatura. O trabalho de investigação clínica permitiu estabelecer uma associação entre a mutação e a apresentação clínica.Conclusão:Em uma população de doentes com hipertrofia ventricular esquerda, documentamos uma prevalência de doença de Fabry de 2,1%. O novo caso foi definido na sequência de uma mutação de significado indeterminado no gene GLA com posterior estudo de patogenicidade. Este estudo permitiu, assim, definir uma nova mutação causal para doença de Fabry - [GLA] c.785G>T; p.W262L (éxon 5).