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Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.
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PMID:Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water. 921

The detection of Legionella sp. in various types of environmental water samples was evaluated using a revised version of the EnviroAmp Legionella kit (Perkin-Elmer), which is based on the polymerase chain reaction (PCR). The new kit employs a modified protocol for the pretreatment of water samples, which is designed to further reduce the inhibitory action of some samples to the PCR. The results of the new kit were compared with those of culture. Seventy-four water samples were examined. Of these samples, 51 were taken from hospital water systems, 16 were household water samples, 5 were from surface water, and 2 were rain water. These samples were examined both by PCR and by culture on MWY Legionella selective agar. Of the 74 samples, 46 (62%) were positive by both test systems. Twenty-five samples (34%) were positive by PCR, but negative by culture. While 3 samples (4%) were negative by both test methods, no sample was negative by PCR and positive by culture. Statistical analysis revealed high positive predictive values of PCR, when L. pneumophila was detected or when the genus Legionella was detected at high intensity. Negative predictive values were high, when the PCR was negative or only weakly positive for the genus Legionella.
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PMID:Evaluation of a new version of the EnviroAmp Legionella kit for the detection of legionellae in water samples by the polymerase chain reaction. 935 40

Modifications to the EnviroAmp Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila, this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila, permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 degrees C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.
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PMID:A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp Legionella system and seroagglutination. 967 68

Using polymerase chain reaction (PCR) for the detection of pathogens that are difficult to grow, such as Legionella species, may reduce difficulties encountered with culture and immunofluorescent staining. We evaluated a commercial PCR and hybridization kit, designed for environmental samples, for the detection of Legionella in respiratory specimens. Sixteen Legionella species cultures tested positive with the Perkin Elmer Legionella EnviroAmp Amplification and Detection kits (Perkin Elmer, Foster City, Calif). The assay detected as few as 100 colony-forming units per milliliter of spiked bronchoalveolar lavage (BAL) fluid, and no false-negative results were obtained. PCR inhibition by blood in the specimens was removed by washing pelleted specimens in sterile distilled water. Of 126 specimens screened with the kit, 1 induced sputum and 3 BAL specimens were positive by PCR. All 4 were validated as true-positive results by culture or serologic testing. The entire PCR and hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 12 days, and serologic conversion may not be demonstrated for weeks. Molecular techniques based on direct extraction and amplification of DNA from respiratory specimens nay be useful for the timely diagnosis of legionellosis.
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PMID:Detection of Legionella by PCR in respiratory specimens using a commercially available kit. 972 3

We have evaluated urine specimens of presumptive cases of legionnaires' disease (110 cases, 173 sample), collected in the past eight years (April, 1990-August, 1998) with the Binax EIA kit which detects the soluble antigen of Legionella pneumophila serogroup (SG) 1, and the Biotest EIA kit which detects Legionella species. Seven cases (19 specimens) were positive for the Binax EIA kit, and nine cases (22 specimens) were positive for the Biotest EIA kit. The sensitivity for culture, PCR, IFA method were 100%, 100%, and 50%, the specificity for these method were 93%, 97.1%, and 90% respectively. Overall agreements for these method were 93.5%, 97.4%, 86.8%, these results suggested that the urinary antigen detection test had high sensitivity and specificity. Our study indicated that concentrated urine samples increase sensitivity. We also evaluated the capabilities of both EIAs to detect soluble antigens were extracted from bacterial suspension of 18 strains of 5 Legionella species by heating. Both assays detected L. pneumophila serogroups 1 to 14, L. bozemanii. The Binax EIA proved to be useful as the Biotest EIA for diagnosis of legionellosis caused by Legionella species and serogroups other than L. pneumophila serogroup 1. Some cases have been shown to excrete antigen for prolonged period of times despite recovery from infection, so that the patient's history should be sought. The urine antigen detection EIA methods proved to be rapid and easy to use, detect antigen in the early stage of the disease with high sensitivity and specificity. Its use for the definition of legionellosis should be considered in Japan.
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PMID:[Evaluation of urinary antigen detection methods for rapid diagnosis of Legionella pneumonia]. 1038 21

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionella species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.
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PMID:Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation. 1079 85

The Binax and the Biotest urinary antigen kits for the detection of Legionnaires' disease caused by organisms other than Legionella pneumophila were compared by testing 45 urine samples from non-Legionella pneumophila serogroup 1 patients previously positive in a broad-spectrum enzyme-linked immunosorbent assay (ELISA). Eighteen were positive with the Binax kit, and 13 were positive with the Biotest. Although neither kit is as sensitive as ELISA, these results extend the number of serogroups and species of Legionella that can be diagnosed with the Binax or Biotest kit.
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PMID:Evaluation of the Binax and Biotest urinary antigen kits for detection of Legionnaires' disease due to multiple serogroups and species of Legionella. 1135 34

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect Legionella DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73 Legionella pneumophila strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other Legionella strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species L. micdadei, L. bozemaniae, and L. dumoffii. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with Legionella culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of Legionella DNA.
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PMID:Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens. 1147 11

The Bartels Legionella Urinary Antigen enzyme immunoassay (Intracel, USA) is intended for the presumptive diagnosis of past or current Legionnaires' disease by qualitative detection of Legionella pneumophila serogroup 1 antigen in human urine. This test was evaluated using single urine samples collected from 349 patients with lower respiratory tract infection of known aetiology. Specificity was estimated as 100% (181 samples, 95% CI: 98%-100%); sensitivity for Legionella pneumophila serogroup 1 was 98.8% (167 samples, 95% CI: 95.7%-99.9%). Assessing assay results using a Visual Interpretation Card provided by the manufacturer in place of a photometer gave rise to one false-positive result among the 78 control samples examined. Providing the endpoint of this assay is determined photometrically, the Bartels Legionella Urinary Antigen enzyme immunoassay appears to be a highly specific and sensitive kit for the diagnosis of infection caused by Legionella pneumophila serogroup 1.
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PMID:Evaluation of the Bartels Legionella Urinary Antigen enzyme immunoassay. 1175 77

OBJECTIVE: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made. METHODS: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested. RESULTS: The PCR was specific for L. pneumophila and no non-Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3-7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure. CONCLUSIONS: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.
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PMID:A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens. 1186 82


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