International Journal of Pediatric Otorhinolaryngology
Identification of Streptococcus pneumoniae and Haemophilus influenzae in culture-negative middle ear fluids from children with acute otitis media by combination of multiplex PCR and multi-locus sequencing typing
Introduction
Acute otitis media (AOM) accounts for 33% of all visits to pediatricians and 40% of all antibiotic use in young children. By three years of age, most children have experienced at least one episode of AOM, and approximately 15%–20% of children suffer from two or more incidents of AOM [1], [2]. Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are two of the major bacterial pathogens that cause AOM in young children [1]. Our laboratories are studying countermeasures against Spn and Hflu infection that might be used to reduce or prevent AOM. Accurate identification of Spn and Hflu in middle ear fluid therefore becomes critical before an effective intervention can be evaluated.
Middle ear fluid (MEF) is the sample recommended for standard bacterial culture for etiologic diagnosis of AOM [1], [3]. For a variety of reasons, however, only approximately 60% of MEF samples from children with AOM are culture-positive for the major bacterial pathogens [3], [25]. It is known that host defense mechanisms and antibiotic treatment prior to collection of MEF specimens may effect pathogen survival and detection [1], [2], [3], [4]. In addition, some bacterial strains, for example optochin-resistant, bile-insoluble, and non-encapsulated Spn strains, cannot be identified by conventional culture methods [5]. Biofilm mode of growth on the middle ear mucosa may be another possible reason for negative cultures of organisms from MEF [26], [27]. During the past decade, polymerase chain reaction (PCR)-based assay methods, including conventional PCR, reverse transcript PCR (RT-PCR), real time PCR, multiplex PCR (mPCR), and others, have been employed to assist etiologic diagnosis of AOM by detecting DNA or RNA of suspected organisms. Although these PCR-based methods have been shown to significantly improve the sensitivity of bacterial detection in AOM [6], [7], [8], [9], false positive results by these methods may occasionally happen. Therefore, the results from PCR-based analysis should be reconfirmed by a secondary method which will provide further detailed information about pathogens before a diagnostic conclusion can be made. Multi-locus sequence typing (MLST) can provide detailed information regarding particular molecular types of the pathogens. The aim of the present study was to improve laboratory methods to accurately detect and identify Spn and Hflu in culture-negative MEF samples from children with AOM using multiplex PCR, followed by MLST to determine molecular types directly from MEF samples.
Section snippets
Patient population, clinical specimens, and bacterial isolation
The children at age of 6–36 months enrolled in this study were recruited from a private pediatric practice, Legacy Pediatrics, in a suburban area of Rochester, New York, from 2003 to 2007. All of the children received standard vaccinations including the 7-Valent Pneumococcal Conjugate Vaccine (Prevnar, Wyeth Pharmaceuticals, Collegeville, PA) at the age appropriate times. Diagnosis of AOM was made by criteria endorsed by the American Academy of Pediatrics (AAP) as previously described [10]. The
Confirmation of Spn and Hflu by mPCR and MLST in culture-positive MEF from children with AOM
In order to test the sensitivity and efficiency of mPCR and MLST, 10 MEF samples that were culture-positive respectively for Spn or Hflu were randomly selected to detect Spn or Hflu by mPCR, and then to determine the sequence types by MLST directly from the MEF without an intermediate culture step. The sequence type data obtained from the MEF samples and their corresponding bacterial culture isolates are shown in Table 2, Table 3, Table 4. The Spn culture-positive MEF samples were all
Discussion
In this study, we have shown that mPCR and MLST results directly from MEF samples matched perfectly (100%) with that of bacterial culture method when MEF samples were culture positive (Table 2). This indicated that the cultured bacterial strains are representative of the in vivo strains when the MEF is culture positive. More importantly, this is the first study, as far as we are aware, to use mPCR and MLST together to detect Spn and Hflu in culture-negative MEF samples from children with AOM.
Conflict of interest statement
The authors have no conflict of interest to declare.
Acknowledgments
This work was funded by the National Institute on Deafness and Other Communication Disorders research grant DC008671, the Thrasher Research Fund award # 02823-2, and an investigator-initiated research grant from Wyeth Pharmaceuticals to M.E.P. The authors gratefully acknowledge the staff of Legacy Pediatrics, Rochester, NY, for their cooperation in sample collection. We also thank Arthur Chang and Safeihkhatoon Moshkani for assistance on PCR, sequence reaction, and data analysis.
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