Avaliação de processos de coleta e transporte de amostras biológicas para diagnósticos de erros inatos do metabolismo em centros de referência

Introdução: A coleta e o transporte de amostras biológicas fazem parte da fase pré- analítica do processo operacional de realização de exames laboratoriais, sendo essa uma fase crítica para as análises. O Serviço de genética Medica (SGM) do Hospital de Clínicas de Porto Alegre (HCPA) é conhecido como um centro de referência nacional e internacional em diagnóstico laboratorial e pesquisa de diferentes doenças genéticas raras, onde são recebidas amostras de todo Brasil e diversos outros países. Devido à importância das avaliações laboratoriais e com o avanço tecnológico na área de diagnósticos verificamos a necessidade de investigação e otimização dos processos de coleta e transporte de amostras biológicas. Objetivo: O principal objetivo desse projeto foi avaliar diferentes métodos de coleta e transporte de amostras biológicas para a realização de testes diagnósticos para erros inatos do metabolismo, assim facilitando o acesso ao diagnóstico, minimizando o número de solicitações de novas amostras, bem como apoiar o desenvolvimento de pesquisas na área. Metodologia: O presente trabalho avaliou 2848 amostras biológicas encaminhadas para o Serviço de Genética Médica do HCPA por 276 instituições, de 25 estados diferentes, verificamos os dados de identificação, volume, temperatura de transporte, tempo decorrido entre a coleta e o transporte em comparação com diferentes sistemas de coleta utilizados pelos serviços associados às redes, entre si e com um sistema padrão desenvolvido pelo nosso serviço. Após uma avaliação inicial realizamos também uma intervenção com material informativo, estratégias educacionais, treinamento de profissionais sobre procedimentos adequados de coleta, remessa e transporte de amostras biológicas. Resultados: 1308 amostras foram enviadas pré-intervenção sendo que destas 254 (19%) apresentaram alguma inconformidade. Após a realização da intervenção através do uso de instruções de coleta e envio uma redução de 62% foi encontrada (119 amostras de 1540). Conclusão: Uma redução considerável no número de amostras inadequadas para uso foi obtida através do uso deste material informativo, tal redução tem considerável impacto nos exames de pesquisa e assistência do HCPA. Este projeto contribuiu para o aperfeiçoamento dos processos de coleta, armazenamento e transporte de material biológico. Protocolos como este podem vir a proporcionar a melhora na qualidade do serviço diagnóstico prestado no SGM e demais utilizadores deste. Além disto, o diagnóstico precoce de uma doença genética poderá facilitar a intervenção mais precoce, através de tratamentos que sabidamente tem um melhor resultado quanto mais cedo se iniciar. Além do mais, o impacto de um diagnóstico preciso e precoce é extremamente benéfico para o sistema de saúde, visando também fortalecer e dar continuidade ao treinamento dos profissionais de saúde e divulgação de informações especializadas.Introduction: The collection and the transport of biological samples belong to the preanalytical phase of laboratory tests, which is a critical step for the analyzes. The Medical Genetics Service (SGM) of Hospital de Clínicas de Porto Alegre (HCPA) is a national and an international reference center for the diagnosis and research of rare genetic diseases, receiving samples from Brazil and several other countries. Due to the importance of laboratory evaluations and technological advances in the area, we verified the need for investigation and optimization of biological sample collection and the transport processes. Objective: The main objective of this project was to evaluate different methods for collecting and transporting biological samples to perform diagnostic tests for inborn errors of metabolism, facilitating access to diagnosis, minimizing the number of requests for new samples, as well as supporting research in the area. Methodology: This study has evaluated 2,848 samples from 276 centers from 25 Brazilian states. We have verified the sample information, the sample volume, temperature of transport, time between sample collection and shipping and we have compared the different collection systems offered by the several networks with a standardized system created by our service. After an initial evaluation prior to any intervention we have also evaluated the efficacy of this novel protocol in order to reduce the need of sample recollection by the use of an informative sheet that lead to the training of health professionals with information regarding sample collection, shipping and transportation of biological samples. Results: 1308 samples were evaluated before the educational intervention, 254 (19%) out of these had at least one nonconformity. After the use of the educational sheet that had information about sample collection and shipping there was a reduction of 11.3% in the preanalytical erros (119 samples out of 1,540). Conclusions: A substantional reduction in the number of inappropriate samples was obtained after the use of this informative material. This reduction has great impact in our health care at the HCPA. This project has contributed to aid sample collection, storage and shipping. Such educational protocols can help to improve the quality of our diagnostic procedures as well as of other services. Besides, early diagnosis of genetic disorders can aid early intervention that is well known to have greater outcomes. Furthermore, the impact of a precise early diagnosis is beneficial to our health system aiming to strengthen the training of health professionals as to spread revelant information

Antimicrobial resistance of enterococci isolated from food in South Brazil : comparing pre-and post-RDC 20/2011

Antimicrobial resistance has been attributed to the overuse of antibiotics. To control the use of antibiotics, Brazil adopted the RDC 20/2011. A comparison the antibiotic-resistance profi le of bacterial has provided important insights into resistance evolution. Enterococci are ubiquitous bacteria recommended to be used as a sentinel organism, in national surveillance systems, for tracking antimicrobial resistance through the food chain. The present study aimed to evaluate the diversity and antimicrobial resistance of enterococci collected from food in South Brazil in 2017 (pos-RDC 20/11) for comparison with isolated in 2007 (pre-RDC 20/11). A total of 310 enterococci were isolated from vegetables and products of animal origin, identifi ed by PCR and MALDI-TOF, tested for antimicrobial susceptibility and screened for resistance genes. Enterococcus casselifl avus was dominant in vegetables and E. faecalis i n products of animal origin. Enterococcal isolates in 2017 were mostly sensitive to ampicillin, gentamicin, chloramphenicol, and vancomycin when compared to isolated collected in 2007. While resistance levels to most compounds remained relatively stable, multidrug resistance decreased by 24% during this period. Our results suggest that RDC 20/11 had a positive outcome in controlling the spread of antimicrobial resistance. This study provides baseline data to measure future changes in the prevalence of resistant enterococci

Phenotype-oriented NGS panels for mucopolysaccharidoses : validation and potential use in the diagnostic flowchart

Mucopolysaccharidosis (MPS) are a group of rare genetic disorders caused by deficiency in the activity of specific lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs). A defect in the activity of these enzymes will result in the abnormal accumulation of GAGs inside the lysosomes of most cells, inducing progressive cellular damage and multiple organ failure. DNA samples from 70 patients with biochemical diagnosis of different MPSs genotypes confirmed by Sanger sequencing were used to evaluate a Next Generation Sequencing (NGS) protocol. Eleven genes related to MPSs were divided into three different panels according to the clinical phenotype. This strategy led to the identification of several pathogenic mutations distributed across all exons of MPSs-related genes. We were able to identify 96% of all gene variants previously identified by Sanger sequencing, showing high sensitivity in detecting different types of mutations. Furthermore, new variants were not identified, representing 100% specificity of the NGS protocol. The use of this NGS approach for genotype identification in MPSs is an attractive option for diagnosis of patients. In addition, the MPS diagnosis workflow could be divided in a two-tier approach: NGS as a first-tier followed by biochemical confirmation as a second-tier

Experience of the NPC Brazil network with a comprehensive program for the screening and diagnosis of Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs

Information and diagnosis networks : tools to improve diagnosis and treatment for patients with rare genetic diseases

Brazil is a country of continental dimensions and most genetic services are concentrated in the Southeast and South, including the Medical Genetics Service of the Hospital de Clínicas de Porto Alegre (MGS/HCPA). As many areas on the country do not have adequate medical genetics support, networks were designed to extend the service of the MGS/HCPA reference center. This paper presents the information and diagnosis networks that have their headquarters at MGS/HCPA: SIAT (National Information System on Teratogenic Agents), SIEM (Information Service on Inborn Errors of Metabolism), Alô Genética (Hello Genetics - Medical Genetics Information Service for Primary Health Care Professionals); Rede MPS Brasil (MPS-Mucopolysaccharidosis Brazil Network); Rede EIM Brasil (IEM-Inborn Errors of Metabolism Brazil Network), Rede NPC Brasil (Niemann-Pick C - NPC Brazil Network), Rede DLD Brasil (LSD-Lysosomal Storage Disorders Brazil Network), Rede DXB (MSUD-Maple Syrup Urine Disease Network), RedeBRIM (Brazilian Network of Reference and Information in Microdeletion Syndromes Project), Rede Neurogenética (Neurogenetics Network), and Rede Brasileira de Câncer Hereditário (Brazilian Hereditary Cancer Network). These tools are very useful to provide access to a qualified information and/or diagnostic service for specialized and non-specialized health services, bypassing difficulties that preclude patients to access reference centers

Biochemical diagnosis of aromatic-L-amino acid decarboxylase deficiency (AADCD) by assay of AADC activity in plasma using liquid chromatography/tandem mass spectrometry

Aromatic l-amino acid decarboxylase (AADC, EC 4.1.1.28) deficiency is a rare genetic disorder characterized by developmental delay, oculogyric crises, autonomic dysfunction and other problems, caused by biallelic mutations in the DDC gene leading to deficient activity of aromatic l-amino acid decarboxylase, an enzyme involved in the formation of important neurotransmitters, such as dopamine and serotonin. A clinical development program of gene therapy for AADC deficiency is ongoing. An important step for the success of this therapy is the early and precise identification of the affected individuals, but it has been estimated that around 90% of the cases remain undiagnosed. The availability measurement of the AADC activity is mandatory for an accurate biochemical diagnosis. Based on these statements, our objectives were to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method suitable for the determination of the AADC activity, and to evaluate its capacity to confirm the deficiency of AADC in potential patients in Brazil. The AADC activities were measured in plasma samples of seven AADC deficient patients and 35 healthy controls, after enzymatic reaction and LC-MS/MS analysis of dopamine, the main reaction product. The results obtained showed clear discrimination between confirmed AADC deficient patients and healthy controls. The method presented here could be incorporated in the IEM laboratories for confirmation of the diagnosis of when a suspicion of AADC deficiency is present due to clinical signs and/or abnormal biomarkers, including when an increased level of 3-O-methyldopa (3-OMD) is found in dried blood spots (DBS) samples from high-risk patients or from newborn screening programs