Pediatr Infect Vaccine.  2015 Aug;22(2):106-112. 10.14776/piv.2015.22.2.106.

Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion

• Byun SW
• Kim HW
• Yoon SH
• Park IH
• Kim KH

• KMID: 2465255
• DOI: http://doi.org/10.14776/piv.2015.22.2.106

Abstract

PURPOSE
The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs.
METHODS
The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs.
RESULTS
The detection limit of the PCR assay was approximately 10(4) colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over 10(6) CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01).
CONCLUSIONS
As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA-specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.

Keyword

Streptococcus pneumoniae; Otitis media with effusion (OME); Loop-mediated isothermal amplification (LAMP); Polymerase chain reaction (PCR); Molecular diagnosis

MeSH Terms

Anti-Bacterial Agents
Child*
Diagnosis*
DNA
Ear, Middle*
Humans
Influenza, Human
Limit of Detection
Otitis Media with Effusion*
Otitis Media*
Otitis*
Pathology, Molecular
Pneumonia
Polymerase Chain Reaction
Sensitivity and Specificity
Stem Cells
Streptococcus pneumoniae*
Streptococcus*
Anti-Bacterial Agents
DNA

Figure

• 

  Fig. 1. Sensitivities of polymerase chain reaction (PCR) and loop-mediated isothermal amplification assay (LAM P) against Streptococcus pneumoniae (SPECGB) spiked in sterile middle ear fluid. P and L indicate PCR and LAMP, respectively, and C (+) and C (—) indicate a positive control for pneumococcal DNA and a negative control, respectively. Bacteria was serially diluted in the middle ear fluid and used for two molecular diagnostic methods. The detection limits of PCR and LAMP are 104 CFU and less than 10 CFU, respectively.

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