ABSTRACT
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.
Subject(s)
Legionella/genetics , Legionella/isolation & purification , Water Pollutants/isolation & purification , Water Supply/analysis , Environmental Monitoring , Netherlands , Polymerase Chain Reaction , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, RNAABSTRACT
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.
Subject(s)
DNA, Bacterial/analysis , Legionella pneumophila/genetics , Legionnaires' Disease/diagnosis , Polymerase Chain Reaction/methods , Adult , Aged , Bacterial Proteins/genetics , DNA, Bacterial/blood , DNA, Bacterial/genetics , Female , Humans , Legionnaires' Disease/blood , Legionnaires' Disease/microbiology , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Peptidylprolyl Isomerase/genetics , Polymerase Chain Reaction/standards , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 5S/genetics , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Bordetella holmesii is a Gram-negative bacterium first identified in 1995. It can cause pertussis-like symptoms in humans. B. holmesii contains insertion sequences IS481 and IS1001, two frequently used targets in the PCR diagnosis of Bordetella pertussis and Bordetella parapertussis infections. To investigate the prevalence of B. holmesii in Finnish and Dutch patients with pertussis-like symptoms and whether B. holmesii has caused any false-positive results in diagnostic PCRs, B. holmesii-specific real-time PCRs were developed. The Finnish methods were conventional IS481 PCR and B. holmesii-specific real-time PCR (LightCycler, Roche) targeting the B. holmesii recA gene. The Dutch methods were IS481 and IS1001 PCRs with conventional or real-time formats and B. holmesii-specific real-time PCR targeting the homologue of IS1001. Of 11,319 nasopharyngeal swabs, 2804 were collected from Finnish patients from 2000 to 2003, and 8515 from Dutch patients from 1992 to 2003. B. holmesii DNA was not found in the samples analysed. The results suggest that B. holmesii is not among the causative agents of pertussis-like symptoms in Finnish and Dutch patients and thus does not in practice confound IS481 and IS1001 PCRs.
Subject(s)
Bordetella/isolation & purification , DNA, Bacterial/genetics , Nasopharynx/microbiology , Whooping Cough/epidemiology , Bacterial Proteins/genetics , Base Sequence , Bordetella/genetics , DNA Transposable Elements/genetics , Finland/epidemiology , Humans , Molecular Sequence Data , Netherlands/epidemiology , Polymerase Chain Reaction/methods , Rec A Recombinases/genetics , Sensitivity and Specificity , Sequence Alignment , Whooping Cough/diagnosisABSTRACT
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.
