RESUMO
The first Spanish multi-centre study on the microbiology of cystic fibrosis (CF) was conducted from 2013 to 2014. The study involved 24 CF units from 17 hospitals, and recruited 341 patients. The aim of this study was to characterise Pseudomonas aeruginosa isolates, 79 of which were recovered from 75 (22%) patients. The study determined the population structure, antibiotic susceptibility profile and genetic background of the strains. Fifty-five percent of the isolates were multi-drug-resistant, and 16% were extensively-drug-resistant. Defective mutS and mutL genes were observed in mutator isolates (15.2%). Considerable genetic diversity was observed by pulsed-field gel electrophoresis (70 patterns) and multi-locus sequence typing (72 sequence types). International epidemic clones were not detected. Fifty-one new and 14 previously described array tube (AT) genotypes were detected by AT technology. This study found a genetically unrelated and highly diverse CF P. aeruginosa population in Spain, not represented by the epidemic clones widely distributed across Europe, with multiple combinations of virulence factors and high antimicrobial resistance rates (except for colistin).
Assuntos
Fibrose Cística/complicações , Farmacorresistência Bacteriana , Variação Genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Adolescente , Adulto , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Espanha/epidemiologia , Virulência , Adulto JovemRESUMO
Introducción. La reacción en cadena de la polimerasa cuantitativa y fluorescente (QF-PCR) identifica, en menos de 24h, las anomalías cromosómicas más frecuentes, buscadas en el cariotipo prenatal convencional: las alteraciones numéricas de los cromosomas 13, 18, 21, X e Y. Introducción. En la QF-PCR se utilizan varios marcadores polimórficos por cromosoma, permitiendo una alta fiabilidad, rapidez, bajo costo y automatización. En cambio, no permite detectar anomalías estructurales, mosaicismos de bajo nivel ni cromosomas marcadores. Objetivo. Desarrollar y valorar un método rápido, practicable y fiable para la detección de aneuploidías de los cromosomas 13,18, 21, X e Y en las muestras recibidas para estudio de cariotipo prenatal en un laboratorio de citogenética. Material y métodos. Además de en los líquidos amnióticos recibidos en el último año (465), la QF-PCR se ha ensayado también, de forma retrospectiva, en las muestras de ADN almacenadas de las enfermedades detectadas en los 2 años anteriores (que corresponden al estudio de unas 1.000 muestras prenatales). Material y métodos. La extracción del ADN se realiza de forma automatizada. Los marcadores utilizados son de alta heterocigosidad y están consensuados internacionalmente (Association for Clinical Cytogenetics. QF-PCR for the diagnosis of aneuploidy best practice guidelines (2007). Disponible en: http://www.cytogenetics.org.uk/prof_standards/professional_standards.htm). El test tiene las mismas condiciones de amplificación para las 4 mezclas multiplex utilizadas. En cada una de ellas se han incluido todos los constituyentes necesarios, excepto la muestra, que se añade en el momento de usarse. Cada amplificado se analiza en el ABI 3130 Genetic Analyzer, de Applied Biosystems. El análisis de los fragmentos obtenidos en la amplificación se realiza siguiendo las recomendaciones publicadas. Resultados. Todos los resultados de las muestras analizadas, normales o patológicas, han coincidido con el resultado del cariotipo. Resultados. En total, se detectaron 12 trisomías 21; 5 trisomías 18; 3 monosomías 45,X; 2 trisomías 13; 2 trisomías 47,XYY; 2 trisomías 47,XXX; 2 trisomías 47,XXY, y 2 triploidías. En todos ellos la QF-PCR propuesta fue diagnóstica. No se detectó una monosomía parcial del cromosoma 18, pero sí pudo ponerse de manifiesto una trisomía parcial del cromosoma 18. Conclusiones. La QF-PCR propuesta es una herramienta útil para el diagnóstico rápido de aneuploidías. Es extremadamente valiosa en el caso de que no crezcan los cultivos de amniocitos, o de contaminación microbiológica de éstos. Sirve para rebajar la ansiedad materna hasta que el cariotipo tradicional esté informado y para tomar decisiones rápidas cuando se encuentran anomalías ecográficas avanzado el segundo trimestre del embarazo (AU)
Introduction. Quantitative fluorescence polymerase chain reaction (QF-PCR) identifies in less than 24h the most common chromosomal anomalies looked for in conventional prenatal karyotyping: numerical alterations of chromosomes 13, 18, 21, X and Y. Introduction. Several polymorphic markers are used in QF-PCR for each chromosome, allowing a high reliability of results, speed, low cost and automation. Nevertheless, QF-PCR can not detect structural abnormalities, low-level mosaicism or marker chromosomes. Objective. To develop and evaluate a fast, reliable and feasible method for detecting aneuploidies of chromosomes 13, 18, 21, X and Y, in the samples received for prenatal karyotyping in a cytogenetics department. Materials and methods. In addition to the 465 amniotic fluids received during last year, stored DNA samples in which a disease was detected in the previous two years were assayed (corresponding to the study of around 1,000 prenatal samples). Materials and methods. DNA extraction is automated. The STR polymorphic markers used, with high heterozygosity, are accepted internationally (Association for Clinical Cytogenetics. QF-PCR for the diagnosis of aneuploidy best practice guidelines (2007). Available at http://www.cytogenetics.org.uk/prof_standards/professional_standards.htm). The test has the same PCR amplification conditions for all four multiplex mixtures used. In each of them All the necessary components were included in each of them, except the sample, which was added at the time of the assay. Each amplified mixture is analyzed into the ABI3130 Genetic Analyzer, Applied Biosystems. Fragments analysis is achieved following the published recommendations. Results. The results of all the tested samples, normal or pathological, matched the outcome of the conventional karyotype. Results. In total the QF-PCR assay detected: 12 trisomy 21; 5 trisomy 18; 3 women 45,X ; 2 trisomy 47,XXY ; 2 trisomy 13 ; 2 Triple XXX ; 2 men XYY; and 2 triploid pregnancies. In each of these cases the proposed QF-PCR was diagnostic. A partial monosomy of chromosome 18 could not be detected, but a partial trisomy of chromosome 18 was identified. Conclusions. The evaluated QF-PCR is a useful tool for the rapid diagnosis of aneuploidies (24h). It is extremely valuable when the amniocyte cultures fail to grow, or suffer microbiological contamination. It serves to reduce maternal anxiety until the conventional karyotype is reported, and to take quick decisions when ultrasound anomalies are found in the second trimester of pregnancy (AU)
