RESUMEN
Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.
Asunto(s)
Legionella/genética , Legionella/aislamiento & purificación , Contaminantes del Agua/aislamiento & purificación , Abastecimiento de Agua/análisis , Monitoreo del Ambiente , Países Bajos , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARNRESUMEN
Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=-0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.
Asunto(s)
ADN Bacteriano/análisis , Legionella pneumophila/genética , Enfermedad de los Legionarios/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Adulto , Anciano , Proteínas Bacterianas/genética , ADN Bacteriano/sangre , ADN Bacteriano/genética , Femenino , Humanos , Enfermedad de los Legionarios/sangre , Enfermedad de los Legionarios/microbiología , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Isomerasa de Peptidilprolil/genética , Reacción en Cadena de la Polimerasa/normas , ARN Ribosómico 16S/genética , ARN Ribosómico 5S/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage. Detection of Legionella DNA in serum can be a valuable tool for the diagnosis of Legionnaires' disease (LD). This report describes two patients with LD diagnosed by PCR using serum samples. In addition, quantification of L. pneumophila DNA using real-time PCR during the course of illness was carried out. The results obtained mirrored both the clinical condition and C-reactive protein values during the course of the illness. Quantification of Legionella DNA in serum using real-time PCR could be a valuable tool to monitor the effects of antimicrobial therapy in patients with LD.