Estudio cualitativo de errores innatos del metabolismo usando resonancia magnética nuclear protónica en un centro de referencia de Bogotá D.C.

ANTECEDENTES: El diagnóstico bioquímico de los Errores Innatos del Metabolismo de molécula pequeña se basa en la detección de metabolitos específicos mediante GC-MS y HPLC. Con base en la capacidad de hacer un análisis más global de los metabolitos, la espectroscopía de RMN (1H-RMN) es una alternativa para un abordaje diagnóstico inicial de estos trastornos. Este trabajo proporciona evidencia sobre perfiles específicos para pacientes con acidemias orgánicas y aminoacidopatías en comparación con sujetos no afectados, usando NMR-400 MHz. MÉTODOS: Se analizaron 36 controles y 17 muestras de orina patológica (180 μL de tampón KH2PO4 1,5M pH 7,0, 540 μL de muestra de orina con estándar interno TMSP-d4 0,5Mm) usando un Bruker Avance III de 400 MHz. Los datos se analizaron usando MESTRENOVA10 y SIMCA P-14. Finalmente, la asignación del cambio químico del metabolito se realizó en comparación con la literatura y las bases de datos como HMDB, BMRB y otros. RESULTADOS: Los análisis de orina en un NMR operado a 400 MHz permitieron una clara discriminación entre individuos sanos y afectados. Se observaron metabolitos característicos para la mayoría de las patologías evaluadas e incluso algunos metabolitos que no son detectados por GC-MS o HPLC como isovaleril-alanina y cetoleucina en IVA y MSUD, respectivamente, que sólo aparecen en el estado agudo de estas enfermedades. Además, se describe el perfil por 1H-RMN de la Deficiencia de Holocarboxilasa Sintetasa, una enfermedad para la cual, en nuestro conocimiento, no hay informes de perfiles específicos por esta técnica. DISCUSIÓN: Estos resultados evidenciaron que la 1H -NMR operada a 400 MHz permitió detectar perfiles altamente sugestivos de EIM e incluso distinguir entre diferentes estados de la enfermedad, aunque en algunos casos el diagnostico no es conclusivo. Con base en nuestras observaciones, consideramos que este análisis puede ser útil para fortalecer la impresión diagnóstica antes de utilizar las técnicas gold standar, lo que permite ahorrar tiempo y dinero; especialmente cuando el diagnóstico diferencial incluye varios grupos de EIM.Convocatoria 06 de 2015 de la Vicerrectoria de Investigación de la Pontificia Universidad Javeriana (convocatoria interna - apoyo a proyectos interdisciplinarios de investigación para el fortalecimiento de grupos de investigación).BACKGROUND: The biochemical diagnosis of IEM involving small molecule metabolism, is based on detection of specific metabolites using GC-MS and HPLC. Based on the capacity to make a more global analysis of metabolites, NMR Spectroscopy (1H-NMR), is an alternative for an initial diagnostic approach of these disorders. This work provides evidence on specifics profiles for patients with organic acidemias and aminoacidopathies compared to unaffected subjects, using NMR-400 MHz. METHODS: 36 controls and 17 pathological urine samples (180 µL KH2PO4 buffer 1,5M pH 7.0; 540 µL of urine sample with internal standard TMSP-d4 0,5Mm) were studied using a 400 MHz Bruker Avance III. Data was analyzed using MESTRENOVA10 and SIMCA P-14. Finally, assignment of metabolite’s chemical shift was done by comparison with literature and databases like HMDB, BMRB and others. RESULTS: NMR-400 MHz, urine analyses allowed clear discrimination between healthy and affected individuals. Characteristic metabolites were observed for most pathologies evaluated and even some metabolites that are not detected by GC-MS or HPLC like isovaleryl-alanine and Ketoleucine in IVA and MSUD, respectively, which only appear in the acute state of these diseases. Additionally, we described the 1H-NMR profile of Holocarboxylase Synthetase Deficiency, a disease for which in our knowledge, there are no reports of 1H-NMR specific profiles. DISCUSSION: Our results evidenced that, although NMR-400 MHz analyses of urine samples were able to detect profiles highly suggestive of IEM and even distinguish among different states of the disease, it may be insufficient to stablish an specific diagnosis in some cases, where 1H-NMR profile may be unconclusive. Based on our observations, we consider that this analyses may be useful to strengthen the diagnostic impression before using gold standards techniques, which results in time and money saving especially when differential diagnosis includes various groups of IEM.Magíster en Ciencias BiológicasMaestrí

Comparación de métodos de microdilución CLSI M27-A2 y EUCAST en aislamientos de Candida spp. en pacientes con cáncer CLSI microdilution M27-A2 and EUCAST method comparison for Candida spp. in patients with cancer

Objetivo: comparar los métodos de referencia de microdilución en caldo de la CLSI M27-A2 y EUCAST, identificando la utilidad y las principales diferencias de cada uno de ellos para los agentes antifúngicos anfotericina B (1), fluconazol (FCZ) e itraconazol (ITZ), contra aislamientos clínicos de Candidaspp. de pacientes con cáncer. Materiales y métodos: se estudiaron 136 aislamientos de C. albicans, 36 de C. tropicalis y 17 de Candidaspp. Se utilizó el índice Kappa ponderado para medir el grado de acuerdo entre los dos métodos. Resultados: se estableció que el grado de concordancia entre los dos métodos para el total de los aislamientos fue alto con AB (&kappa;: 1) y FCZ (&kappa;: 0.74) y bajo al utilizar ITZ (&kappa;: 0.49). La concordancia fue variable y especie-específica: para ITZ y FCZ en C. albicans fue de 0,45 y 0,64; en C. tropicalis, de 0,48 y 0,91; y en Candidaspp. de 0,73 y 0,87, respectivamente. Discusión: este estudio sugiere que las pruebas de sensibilidad antifúngica para los dos métodos son equivalentes en lo esencial. Deben considerarse las diferencias y discrepancias asociadas a la especie implicada, el tipo de antifúngico utilizado y los tiempos de incubación, que puede producir variaciones al interpretar los resultados obtenidos de acuerdo con la metodología empleada.<br>Objective: compare the broth microdilution testing reference standards CLSI M27-A2 and EUCAST, identifying the usefulness of each one of them and their main differences, against the antifungal agents amphotericin B (1), fluconazole (FCZ), and itraconazole (ITZ) using clinical isolates of Candidaspp. in cancer patients. Methods: isolates of C. albicans (n=136), C. tropicalis (n=36), and Candidaspp. (n=17) were tested by the two methods. The Kappa index was used to establish the degree of agreement between the methods. Results: the degree of agreement between the two methods was high for AB (&kappa;: 1) and FCZ (&kappa;: 0.74) and was low for ITZ (&kappa;: 0.49). Agreement was variable and specific for the various species: for ITZ and FCZ in C. albicans, it was 0.45 and 0.64, respectively. In C. tropicalis, it was of 0.48 and 0.91, and in Candidaspp., it was 0.73 and 0.87 respectively. Discussion: this study suggests that antifungal susceptibility testing using both methods is equivalent. Attention should be focused on differences and discrepancies associated with the species tested, the type of antifungal agent, and the incubation times, which can cause variations at the moment of interpreting the results obtained

1H-nuclear magnetic resonance analysis of urine as diagnostic tool for organic acidemias and aminoacidopathies

The utility of low-resolution H-NMR analysis for the identification of biomarkers provided evidence for rapid biochemical diagnoses of organic acidemia and aminoacidopathy. H-NMR, with a sensitivity expected for a field strength of 400 MHz at 64 scans was used to establish the metabolomic urine sample profiles of an infant population diagnosed with small molecule Inborn Errors of Metabolism (smIEM) compared to unaffected individuals. A qualitative differentiation of the H-NMR spectral profiles of urine samples obtained from individuals affected by different organic acidemias and aminoacidopathies was achieved in combination with GC-MS. The smIEM disorders investigated in this study included phenylalanine metabolism; isovaleric, propionic, 3-methylglutaconicm and glutaric type I acidemia; and deficiencies in medium chain acyl-coenzyme and holocarboxylase synthase. The observed metabolites were comparable and similar to those reported in the literature, as well as to those detected with higher-resolution NMR. In this study, diagnostic marker metabolites were identified for the smIEM disorders. In some cases, changes in metabolite profiles differentiated post-treatments and follow-ups while allowing for the establishment of different clinical states of a biochemical disorder. In addition, for the first time, a H-NMR-based biomarker profile was established for holocarboxylase synthase deficiency spectrum

Research, diagnosis and education in inborn errors of metabolism in Colombia: 20 years’ experience from a reference center

Abstract The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system