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Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

ABSTRACT: A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products. The specificity of the reaction was verified with various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods. A detection level of 10 bacterial cells/reaction for each of the study organisms was achieved. By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms. In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCR negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms. Thus, the multiplex PCR method improves the detection rate significantly compared to that of the conventional culture method.

SUBMITTER: Hendolin PH

PROVIDER: S-EPMC230074 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

Hendolin P H PH Markkanen A A Ylikoski J J Wahlfors J J JJ

Journal of clinical microbiology 19971101 11

A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplific ...[more]

PMID: 9350746

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Project description:Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

Project description:BackgroundBecause previous studies have indicated that otitis media may be a polymicrobial disease, we prospectively analyzed middle ear effusions of children undergoing tympanostomy tube placement with multiplex polymerase chain reaction for four otopathogens.MethodsMiddle ear effusions from 207 children undergoing routine tympanostomy tube placement were collected and were classified by the surgeon as acute otitis media (AOM) for purulent effusions and as otitis media with effusion (OME) for non-purulent effusions. DNA was isolated from these samples and analyzed with multiplex polymerase chain reaction for Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis.Results119 (57%) of 207 patients were PCR positive for at least one of these four organisms. 36 (30%) of the positive samples indicated the presence of more than one bacterial species. Patient samples were further separated into 2 groups based on clinical presentation at the time of surgery. Samples were categorized as acute otitis media (AOM) if pus was observed behind the tympanic membrane. If no pus was present, samples were categorized as otitis media with effusion (OME). Bacteria were identified in most of the children with AOM (87%) and half the children with OME (51%, p < 0.001). A single bacterial organism was detected in middle ear effusions from children with AOM more often than those with OME (74% versus 33%, p < 0.001). Haemophilus influenzae was the predominant single organism and caused 58% of all AOM in this study. Alloiococcus otitidis and Moraxella catarrhalis were more frequently identified in middle ear effusions than Streptococcus pneumoniae.ConclusionsHaemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis were identified in the middle ear effusions of some patients with otitis media. Overall, we found AOM is predominantly a single organism infection and most commonly from Haemophilus influenzae. In contrast, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.

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Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

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Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

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