Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Xu, Jianguo; Millar, B. Cherie; Moore, John E.; Murphy, Karen; Webb, Hugh; Fox, Andrew; Cafferkey, Mary T.; Crowe, M.

doi:10.1046/j.1365-2672.2003.01839.x

Cited by 66 publications

(42 citation statements)

References 25 publications

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“…The PCR is one of the most sensitive of such methods. Most PCR-based identifications in current clinical use rely on amplification of conserved genes, such as those encoding elongation factors (20) or RNA polymerase (rpoB) (6), or ribosomal DNA genes (10,26), followed by detection of species-specific sequences in the product (12,13). In some cases, species-specific genes, such as those encoding cytotoxins (4), can be amplified at the outset.…”

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confidence: 99%

“…In some cases, species-specific genes, such as those encoding cytotoxins (4), can be amplified at the outset. Enhanced strategies include the use of multiplexing (14,26) or of highly parallel techniques, such as diagnostic DNA microarrays (5,8), to amplify and detect multiple sequences at once. Since each PCR primer can be considered a separate reagent, the quality control issues of such testing become more formidable with each additional gene target.…”

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confidence: 99%

“…Most PCR-based bacterial identifications performed in the routine clinical laboratory, however, still require nucleic acids obtained from isolated colonies. Direct PCR of clinical samples is usually restricted to detecting a small number of species and to a specific sample (typically a normally sterile body fluid, such as cerebrospinal fluid or plasma) (14,26), which must be extracted in such a way as to preserve nucleic acids while removing PCR inhibitors.…”

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confidence: 99%

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Comparison of Two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Identification of Bacteria to the Species Level

Cherkaoui¹,

Hibbs

Emonet³

et al. 2010

J Clin Microbiol

453

296

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Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Comparison of Two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Identification of Bacteria to the Species Level

Cherkaoui¹,

Hibbs

Emonet³

et al. 2010

J Clin Microbiol

453

296

View full text Add to dashboard Cite

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“…Molecular techniques overcome some of the limitations of conventional culture under several clinical conditions (5)(6)(7)(8)(9)(10). Nevertheless, the role of molecular techniques in the diagnosis of VAP-RBSI remains unclear, and available approaches have not been extensively tested in the routine of a clinical microbiology laboratory.…”

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confidence: 99%

Use of Universal 16S rRNA Gene PCR as a Diagnostic Tool for Venous Access Port-Related Bloodstream Infections

et al. 2013

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Amplification of the universal 16S rRNA gene using PCR has improved the diagnostic yield of microbiological samples. However, no data have been reported on the reliability of this technique with venous access ports (VAPs). We assessed the utility of 16S rRNA PCR for the prediction of VAP-related bloodstream infection (VAP-RBSI). During a 2-year period, we prospectively received all VAPs removed by interventional radiologists. PCR and conventional cultures were performed using samples from the different VAP sites. We compared the results of PCR with those of conventional culture for patients with confirmed VAP-RBSI. We collected 219 VAPs from 219 patients. Conventional VAP culture revealed 15 episodes of VAP-RBSI. PCR revealed a further 4 episodes in patients undergoing antibiotic therapy which would have gone undetected using conventional culture. Moreover, it had a negative predictive value of 97.8% for the prediction of VAP-RBSI when it was performed using biofilm from the internal surface of the port. In conclusion, universal 16S rRNA PCR performed with samples from the inside of VAPs proved to be a useful tool for the diagnosis of VAP-RBSI. It increased detection of VAP-RBSI episodes by 21.1% in patients undergoing antibiotic therapy whose episodes would have gone undetected using conventional culture. Therefore, we propose a new application of 16S rRNA PCR as a useful tool for the diagnosis of VAP-RBSI in patients receiving antibiotic therapy. C onfirmation of colonization of venous access ports (VAPs) requires culture of both port reservoir contents and the catheter tip (1). However, conventional culture of VAPs could prove negative when antibiotic therapy is prescribed before catheter withdrawal. Therefore, an episode of VAP-related bloodstream infection (VAP-RBSI) might not be confirmed, as diagnosis requires the isolation of the same microorganism in blood culture as in the colonized VAP (2-4).Molecular techniques overcome some of the limitations of conventional culture under several clinical conditions (5-10). Nevertheless, the role of molecular techniques in the diagnosis of VAP-RBSI remains unclear, and available approaches have not been extensively tested in the routine of a clinical microbiology laboratory.Our main objective was to assess the validity values of 16S rRNA PCR to analyze VAPs from patients with confirmed VAP-RBSI. MATERIALS AND METHODSSetting. Ours was a prospective study performed between July 2009 and April 2011 at a large institution in Madrid, Spain.We included all tunneled VAPs (Port-A-Caths) that were routinely removed in the Department of Vascular Interventional Radiology, irrespective of the reason for withdrawal. We also included those devices with suspicion of infection that were removed in surgery departments or emergency rooms.We excluded VAPs that were colonized exclusively by fungi. Laboratory procedures. When the VAPs arrived at the microbiology laboratory, we performed culture and broad-range real-time 16S rRNA gene PCR followed by direct sequencing using port content ...

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“…Schuurman et al [17], found broad range PCR was more useful in meningitis with sensitivity of 86%, specificity of 97%, positive predictive value of 80% and negative predictive value of 98% as compared to conventional culture. Xu et al [19], recommended use of highly resolving polyacrylamide matrix for separation of amplicons and digestion fragments. Thus, universal PCR followed by RFLP is an alternative, rapid, simple and effective method for detection of bacterial pathogens in CSF.…”

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confidence: 99%

Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

Kalghatgi

Praharaj

Sahni³

et al. 2008

Medical Journal Armed Forces India

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Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Cited by 66 publications

References 25 publications

Comparison of Two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Identification of Bacteria to the Species Level

Comparison of Two Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Methods with Conventional Phenotypic Identification for Routine Identification of Bacteria to the Species Level

Use of Universal 16S rRNA Gene PCR as a Diagnostic Tool for Venous Access Port-Related Bloodstream Infections

Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

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