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

Abstract

Objective

Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are major etiologic pathogens for acute otitis media (AOM). However, when Spn and Hflu strains are not identified by traditional culture methods, use of alternative PCR-based diagnosis becomes critical. This study aimed to develop a combined molecular method to accurately detect these otopathogens.

Methods

Middle ear fluid (MEF) samples were collected by tympanocentesis from children with AOM to isolate Spn and Hflu by standard culture procedures. Multiplex PCR (mPCR) and multi-locus sequence typing (MLST) techniques were used to detect Spn and Hflu in culture-negative MEF samples.

Results

We found 20 Spn or Hflu culture-positive MEF samples that were mPCR-positive and typeable by MLST. The sequences of the housekeeping genes and the MLST allelic profiles obtained from Spn or Hflu culture isolates matched exactly MEF samples that were tested directly without culture isolation. Of 63 MEF samples that were culture-negative for Spn, 38% (24/63) were mPCR-positive for Spn. Of 50 MEF samples that were culture-negative for Hflu, 24% (12/50) were mPCR-positive for Hflu. Among these culture-negative but mPCR-positive MEF samples, 25% (6/24) and 25% (3/12) were typeable by MLST for Spn and Hflu, respectively.

Conclusions

MEF samples may be analyzed with mPCR and MLST directly without culture isolation and the addition of mPCR and MLST may accurately identify Spn and Hflu in MEF of children with AOM when bacterial culture is negative.

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.