Using a multiplex PCR to simultaneously detect four bacterial otopathogens in the middle ear effusions obtained from children undergoing routine tympanostomy tube placement, we found distinct bacterial profiles for AOM and 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 patients 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.
Overall, we found AOM is predominantly a single organism infection, whereas, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.
H. influenzae was the predominant bacterial species identified, comprising 66% (25 of 38) of middle ear effusions with AOM and 24% (40 of 169) of middle ear effusions with OME. We observed a higher proportion of samples with H. influenzae and a lower proportion with S. pneumoniae than what has been previously reported. Kaur et al., using a similar multiplex PCR approach on AOM middle ear effusion, detected H. influenzae in 31% of children who were on antibiotic prior to sample acquisition and 39% in children who had not undergone antibiotic treatment . However, Kaur et al. utilized specifically culture-negative MEF, a distinction we have not made with the samples obtained for our research. In other work using culture-negative AOM MEE samples, Xu et al. observed an H. influenzae percentage of 24% .
While the levels of H. influenzae in our patients appear to be higher than what others have seen, we observed the opposing trend with respect to S. pneumoniae. S. pneumoniae, regarded as one of the 3 most prevalent bacterial contributors to OM infection, was identified in only 2 of 38 AOM patients (~5%) and 8 of 169 OME patients (~5%). In the studies listed above by Kaur et al. and Xu et al., S. pneumoniae was identified in around 38%–57% of culture-negative AOM MEF samples, percentages that are considerably higher than what was observed in our study [13, 21].
A number of factors may explain our finding that S. pneumoniae was detected in only 5% of all study subjects. Published studies of pneumococcal prevalence in otitis media report a wide incidence range. Brooke et al. used standard culture based techniques to detect S. pneumoniae in approximately 16% of OME effusions . Post et al. used PCR to detect S. pneumoniae in OME effusions at a higher percentage of approximately 30% . Research conducted by Hendolin et al. detected S. pneumoniae in only 8% of cases examined . We used the same multiplex PCR primers used by Hendolin et al. Kaur et al. used these PCR primers to identify S. pneumoniae in approximately 57% of effusions from children with AOM , and this data does not suggest that our PCR assay has a low sensitivity for detection of pneumococcus.
The low S. pneumoniae incidence found in our study might be explained by the effectiveness of pneumococcal conjugate vaccination. A heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in 2000, and since its acceptance for widespread use there has been a shift in the incidence of otitis media pathogens. In the years immediately following PCV7 introduction, H. influenzae emerged as the most common AOM isolate [14, 24]. More recently, S. pneumoniae serotypes not included in the PCV7 vaccine have been increasingly isolated from AOM cases . In 2010, a new modified pneumococcal conjugate vaccine was introduced to combat the emergence of S. pneumoniae serotypes not included in the original PCV7 vaccine. It contained components from 13 serotypes of S. pneumoniae (7 serotypes included in the PCV7 vaccine along with 6 recently emerging serotypes). Our findings are consistent with improved pneumococcal vaccine prevention in children enrolled in this study.
Another interesting result was the high incidence of A. otitidis in our OME samples (approximately 25%, single and co-infections included). A. otitidis was first identified in OME samples by Faden et al. . It is a Gram-positive organism that exhibits very slow growth, making it very difficult to identify through standard culture techniques. It has been thought of as solely an OME pathogen; however, it is increasingly recognized as a pathogen in AOM [13, 25, 26]. Our findings support what has been reported previously in OME cases . The A. otitidis results further demonstrate the importance of using methods other than standard culture for the identification of fastidious otopathogens.