Rapid detection of Legionella species in bronchoalveolar lavage fluids with the EnviroAmp Legionella PCR amplification and detection kit (original) (raw)
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Medical Microbiology and Immunology, 2002
Molecular assays for qualitative detection of Legionella spp. in clinical specimens were evaluated. DNA extraction was done either with a fully automated DNA extraction protocol on the MagNA Pure LC System or with manual DNA extraction. Amplification and detection were done by real-time polymerase chain reaction (PCR) on the LightCycler (LC) instrument. Oligonucleotides were derived from the 16S rRNA gene of Legionella spp. The assays included a specially designed DNA fragment as Legionella-specific internal control. For both molecular assays, the detection limit was determined to be 5 CFU per LC PCR run. Sixty-one clinical specimens were tested with the molecular assays. Results were compared to culture. Five samples were found to be positive with the molecular assays. Three of them were positive in culture. No inhibition was found throughout the whole study. In conclusion, the molecular assays described may lead to safe and early diagnosis of Legionnaires' disease. They proved to be suitable for the routine molecular diagnostics laboratory.
Folia Microbiologica, 2000
Polymerase chain reaction (PCR) was used for detecting Legionella DNA in water, sputum, tracheal aspirate and bronchoalveolar lavage fluid. There is paucity of data on the use of PCR for detection of Legionella in serum and urine samples. In 82 patients admitted with community-acquired pneumonia, urinary PCR was used in addition to urinary antigen assay for Legionella pneumophila serogroup 1 and serological tests (indirect immunofluorescence and ELISA) in paired sera. PCR was positive in urine samples from 21 patients (26 %): in six of seven patients with acute legionellosis by CDC criteria, and 15 patients with negative urine antigen showing no fourfold rise in antibody titers in immunofluorescence test.
Detection and quantification of Legionella pneumophila in water samples using competitive PCR
Canadian Journal of Microbiology, 2006
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.
Diagnostic Microbiology and Infectious Disease, 2008
Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman® real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colonyforming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3 % (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.
Quantitative real-time PCR tests for diagnostic and prognostic purposes in cases of legionellosis
Clinical Microbiology and Infection, 2010
The usefulness of two quantitative real-time PCR assays (qrt-PCRmip targeting Legionella pneumophila, and qrt-PCR16S targeting all Legionella species) performed on lower respiratory tract (LRT) samples for diagnostic and prognostic purposes in 311 patients hospitalized for community-acquired pneumonia (CAP) in Rhô ne-Alpes (France) was evaluated. The Now Legionella urinary antigen test (UAT) from Binax (Portland, ME, USA) was used as a reference test. Samples were divided into two groups. Group A included 255 CAP patients admitted to Chambery hospital in 2005 and 2006. The Now Legionella UAT was positive in 14 patients. Sensitivities, specificities, positive predictive and negative predictive values for both qrt-PCR tests were 63.6, 98.7, 77.7 and 97.4%, respectively. Group B included 56 consecutive legionellosis patients diagnosed during a 4-year period (2003-2006) at the Grenoble University Hospital. The qrt-PCR16S and qrt-PCRmip displayed a sensitivity of 82.14 and 80.4%, respectively. Among the 70 legionellosis cases, L. pneumophila serogroup 1 was isolated in 15;
Annals of agricultural and environmental medicine : AAEM, 2008
The aim of this work was to evaluate three currently available isolation methods for Legionella using water samples and swabs of a single pediatric hospital water system. Additionally, high risk patients were screened for the presence of Legionella pneumophila antigen in urine. Fifteen water samples and 11 swab samples were collected from distal sites at 18 sampling locations. The International Standard Method (PN-ISO11731-2) based on membrane filtration and direct culture of bacteria on selective media were compared with amoebic co-culture. The numbers of legionellae detected exceeded 10(2) cfu/100 ml in 50% of the samples. All the positive samples contained L. pneumophila SGs 2-14. Urine samples were obtained from 57 immunosuppressed children and screened for the presence of L. pneumophila serogroup (SG) 1 antigen by Legionella urinary antigen EIA. Of the 57 urine samples tested for the presence of Legionella pneumophila SG 1 antigen, none were positive. Our results highlight the ...
Investigation of Legionella pneumophila and other Legionella species in atypical pneumonia patients
Türk hijiyen ve deneysel biyoloji dergisi, 2022
Investigation of Legionella pneumophila and other Legionella species in atypical pneumonia patients Atipik pnömonili hastalarda Legionella pneumophila ve diğer Legionella türlerinin araştırılması Kerim PARLAK 1 (ID), Ayşegül GÖZALAN 2 (ID), Sibel AYDOĞAN 3 (ID), Adem KOYUNCU 4 (ID), Hatice Canan HASANOĞLU 5 (ID), Selin NAR ÖTGÜN 6 (ID), Ziya Cibali AÇIKGÖZ 7 (ID) ÖZET Amaç: Bu çalışmada atipik pnömoni tanısı alan 50 hastada kültür, üriner antijen testi ve moleküler yöntemler kullanarak Legionella türlerinin araştırılması amaçlanmıştır. Yöntem: Solunum yolu örneklerinden Legionella türlerinin izolasyonu için seçici olmayan BCYE-α (Oxoid, İngiltere) ve seçici BMPA (Oxoid, İngiltere) besiyerleri kullanılmıştır. İdrar örneklerinde L. pneumophila serogrup 1'e özgü bakteriyel antijenin varlığı Alere BinaxNOW Legionella Üriner Antijen Kart (Abbott, ABD) testi ile araştırılmıştır.Tüm solunum yolu örnekleri Duplicα RealTime Legionella pneumophila 23S rRNA spesifik bölgesini saptayan (Euroclone Diagnostica, İtalya) ticari kit ve iki laboratuvar yapımı PCR yöntemi ile test edilmiştir. Laboratuvar yapımı jel elektroforez PCR testinde Legionella spp. için 16S ribozomal RNA gen kısmi dizilerinden tasarlanan Leg primerleri ve L. pneumophila için Lmip (macrophage infectivity potentiator) genini hedefleyen primerler ABSTRACT Objective: The aim of this study is to investigate Legionella species in 50 patients with atypical pneumonia, using culture, urinary antigen test and molecular techniques. Methods: Non-selective BCYE-α media (Oxoid, England) and selective BMPA media (Oxoid, England) were used to isolate Legionella spp. from respiratory tract samples. The urinary samples of the patients were tested with the Alere BinaxNOW Legionella Urinary Antigen Card (Abbott, US) test to identify the presence of L. pneumophila serogroup 1 specific bacterial antigen. All respiratory tractsamples were tested with a commercial Duplicα RealTime Legionella pneumophila 23S rRNA specific region detection kit (Euroclone Diagnostica, Italy) and two home-made PCR methods. Home-made gel electrophoresis PCR tests were performed using Leg primers designed from 16S ribosomal RNA gene partial sequences for Legionella spp and primers targeting the Lmip (macrophage