Figures
Abstract
Otitis media is a prominent disease among children. Previous literature indicates that otitis media is a polymicrobial disease, with Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis and Moraxella catarrhalis being the most commonly associated bacterial pathogens. Recent literature suggests that introduction of pneumococcal conjugate vaccines has had an effect on the etiology of otitis media. Using a multiplex PCR procedure, we sought to investigate the presence of the aforementioned bacterial pathogens in middle ear fluid collected from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center during the period between January 2011 and March 2014. In purulent effusions, one or more bacterial organisms were detected in ~90% of samples. Most often the presence of H. influenzae alone was detected in purulent effusions (32%; 10 of 31). In non-purulent effusions, the most prevalent organism detected was A. otitidis (26%; 63 of 245). Half of the non-purulent effusions had none of these otopathogens detected. In purulent and non-purulent effusions, the overall presence of S. pneumoniae was lower (19%; 6 of 31, and 4%; 9 of 245, respectively) than that of the other pathogens being identified. The ratio of the percentage of each otopathogen identified in purulent vs. non-purulent effusions was >1 for the classic otopathogens but not for A. otitidis.
Citation: Holder RC, Kirse DJ, Evans AK, Whigham AS, Peters TR, Poehling KA, et al. (2015) Otopathogens Detected in Middle Ear Fluid Obtained during Tympanostomy Tube Insertion: Contrasting Purulent and Non-Purulent Effusions. PLoS ONE 10(6): e0128606. https://doi.org/10.1371/journal.pone.0128606
Academic Editor: Bernard Beall, Centers for Disease Control & Prevention, UNITED STATES
Received: May 12, 2014; Accepted: April 28, 2015; Published: June 3, 2015
Copyright: © 2015 Holder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This project was supported by R01 DC10051 from the National Institutes of Health to WES, T32 AI007401 from the National Institutes of Health to Dr. Griffith D. Parks (PI), and 11GRNT7980017 from American Heart Association to SDR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Co-author Sean Reid is a PLOS ONE Academic Editor. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.
Introduction
Otitis media (OM) is a leading cause for outpatient visits as well as antibiotic prescriptions for children[1–5]. It is estimated that by 3 years of age 80% of children will have experienced at least one case of OM[6, 7], and 40% of these children will experience six or more recurrences by 7 years of age[8]. Annual direct costs associated with OM are estimated to approach $3–5 billion[9–11], and indirect costs which include loss of productivity and lost working days by family members drive this value higher[12, 13].
Distinct from our previous work[14], this study focuses on the prevalence of four otopathogens (Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis) in middle ear fluid collected from children undergoing tympanostomy tube placement at Wake Forest Baptist Medical Center (Winston-Salem, NC, USA) from January 2011 until March 2014, after the introduction of the pneumococcal conjugate vaccine PCV13.
Materials and Methods
Subjects and Sample Collection
Children who had tympanostomy tube placement at Wake Forest Baptist Medical Center from January 2011 through March 2014 and had middle ear fluid at the time of surgery were eligible. Discarded samples of middle ear effusions were evaluated for bacterial pathogens, as described below.
Study specimens were collected from middle ears that had effusions at the time of tympanostomy tube placement. Fluid was aspirated into a sterile trap. Specimens were categorized as purulent or non-purulent fluid behind the tympanic membrane by the otolaryngologist at the time of tympanostomy tube placement. Fluids visually observed by the pediatric otolaryngologist to have a whitish appearance with a milky or mucoid consistency were designated as purulent. All middle ear effusion samples were kept at room temperature and transported to the research laboratory within 2 hours and then refrigerated at 4°C until DNA isolation. Samples were processed within 5 days of refrigeration.
Isolation of DNA
DNA was extracted from middle ear effusions as described previously[14]. Briefly, middle ear effusions were incubated with various degradation components (mutanolysin, lysozyme, sodium dodecyl sulfate (SDS), RNase, proteinase K) to disrupt bacterial cell walls, remove RNA, and degrade proteins. Remaining DNA was phenol:chloroform:isoamyl alcohol extracted and ethanol precipitated. Resuspended DNA precipitates were used as template material in the multiplex PCR procedure.
Polymerase Chain Reaction
A multiplex PCR procedure created by Hendolin et al[15] was used with minor modification to simultaneously detect H. influenzae, S. pneumoniae, A. otitidis, and M. catarrhalis in middle ear effusions, as previously described[14]. The modification mentioned above was that the polymerase used in the procedure (5 PRIME HotMasterMix) required an extension temperature of 65°C rather than the often used 72°C associated with Taq polymerase.
Chart Review
This study was reviewed and approved by the Wake Forest School of Medicine Institutional Review Board. Sex, age, medications, clinical characteristics and prior antimicrobial treatments were collected from the subject record without identifiable information. Although identifiable information was not collected, administrative data reveals that the average age of children who had tympanostomy tubes placed during this study period was 2.7 years (range of 0–18 years). The demographic characteristics, current medications, previous antibiotic use, and past medical history were all determined by medical record review using a standardized clinical form. The medical record typically contained notes about ear infections from the primary care provider as well as clinic visits to the otolaryngologist.
Analyses
We compared the demographic characteristics, clinical characteristics, or microbiologic results of children with purulent and non-purulent middle ear effusions by using chi-square analyses or Fisher's exact tests. For children with bilateral middle ear effusions, the results from each ear were combined so that each child had one result. The unadjusted odds ratio and 95% confidence intervals were calculated for each variable. A multivariate logistic regression analysis was performed to compute the odds of a purulent infection as compared to a non-purulent infection for each of four otopathogens and age groups. We lacked sufficient power to analyze other variables. STATA 12.1 (College Station, TX) was used for all statistical analyses.
Results
A total of 276 children had middle ear fluid collected at the time of tympanostomy tube placement from January 2011 through March 2014 (Fig 1). Half were 1–3 years of age, two-thirds were male, and 58% were White (Table 1). Children with purulent effusions were more likely to be 1–3 years of age, to have a history of ear infections, to have been on antibiotics within the past 6 months, and to be on antibiotics at the time of surgery than children with non-purulent effusions.
Overall, 149 (54%) of 276 children had middle ear fluid that were PCR positive for the presence of H. influenzae, S. pneumoniae, A. otitidis, and/or M. catarrhalis. Of these 149 PCR positive samples, 117 (79%) identified a single organism and 32 (21%) were polymicrobial (Fig 1).
The three classic otopathogens were more likely to be identified as either a single organism or a polymicrobial component in purulent than non-purulent effusions (Tables 2 and 3). This pattern was observed for H. influenzae (52% vs. 18%, p<0.001), S. pneumoniae (19% vs. 4%, p<0.001) and M. catarrhalis (26% vs. 12%, p = 0.04) but not for A. otitidis (23% vs. 26%, p = 0.71).
A single organism was identified in 61% percent (19 of 31) of all purulent effusions (Table 2), most frequently H. influenzae followed by M. catarrhalis. S. pneumoniae and A. otitidis were identified as single organisms in a minority of purulent effusions. All four otopathogens contributed to the 26% of polymicrobial purulent effusions.
A single organism was identified in 40% (98 of 245) of non-purulent effusions (Table 2). A. otitidis accounted for almost half the single organisms identified and H. influenzae accounted for about a third, with S. pneumoniae and M. catarrhalis being identified less frequently. Overall, 10% of non-purulent effusions were polymicrobial, containing H. influenzae, A. otitidis, and M. catarrhalis with similar frequency and infrequently harboring S. pneumoniae.
H. influenzae was identified 2.9x more frequently in purulent effusions than non-purulent effusions (52% vs. 18%); S. pneumoniae was identified 4.8x more frequently in purulent effusions than non-purulent effusions (19% vs. 4%); M. catarrhalis was identified 2.2x more frequently in purulent effusions than non-purulent effusions (26% vs. 12%). In contrast, A. otitidis was present in relatively equal amounts in purulent effusions and non-purulent effusions (23% vs. 26%) (Table 3) In adjusted and unadjusted multiple logistic regression analyses (Table 4), predictors of purulent as opposed to non-purulent middle ear fluid were H. influenzae, S. pneumoniae, and age <1 year.
Discussion
This prospective study evaluated the presence of four otopathogens (H. influenzae, S. pneumoniae, A. otitidis, and M. catarrhalis) in middle ear fluids obtained from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center from January 2011 through March 2014. Our major findings were: (1) analyses of purulent effusions most often yielded the identification of a single bacterial organism rather than a polymicrobial mixture; (2) the most prevalent single organism identified in purulent effusions was H. influenzae; (3) ~90% of purulent effusions indicated the presence of one or more of the otopathogens; (4) A. otitidis was the most common organism identified in non-purulent effusions; (5) half of the non-purulent effusion samples indicated the absence of all of the otopathogens being evaluated; (6) the overall prevalence of S. pneumoniae in effusion samples was low; (7) H. influenzae, S. pneumoniae, and M. catarrhalis all occurred ≥2-fold more prevalently in purulent effusions than non-purulent effusions; however, A. otitidis occurred equally among purulent and non-purulent effusions.
Our results obtained with purulent effusions correlate with a potentially developing trend in AOM etiology. Historically, the bacterial organisms most commonly identified as AOM pathogens have been S. pneumoniae, H. influenzae, and M. catarrhalis[16–19]. After introduction of the heptavalent pneumococcal conjugate vaccine PCV7 in 2000, H. influenzae briefly became the most prevalent otopathogen identified in cases of OM[20, 21]. Shortly thereafter, serotypes of S. pneumoniae not covered in the initial conjugate vaccine design became prevalent in OM cases and were identified in proportions equaling that of H. influenzae[22–24]. In 2010, a new vaccine incorporating PCV7 S. pneumoniae serotypes as well as 6 new emerging serotypes was released for widespread usage in the United States. Recent data based on results from Rochester, NY detailing the prevalence of bacteria causing AOM in North America in 2012 suggest that a shift in OM bacterial etiology may again be occurring[25]. Briefly, H. influenzae has reemerged as the predominant AOM pathogen, followed by M. catarrhalis. The prevalence of S. pneumoniae in cases of AOM has decreased to levels below those of the other two common otopathogens. The purulent effusion data obtained in our study revealing high levels of H. influenzae and M. catarrhalis in conjunction with low levels of S. pneumoniae are similar to the results described by Casey and Pichichero[25] and Zhao et al[26]. Notably, our subject population underwent tympanostomy tube placement for clinical indications so that most would have recurrent or chronic OM infection and may differ from studies of children from outpatient pediatric clinics, which could include first episodes of otitis media. Also, our designation of purulence based on visual subjectivity rather than molecular quantification represents a potential limitation to this study.
A. otitidis is a slow-growing organism that has been historically overlooked as an OM pathogen due to the use of standard bacterial culture techniques when evaluating organisms present in OM middle ear fluid. Because A. otitidis grows poorly in standard culture, the increasing use of PCR to identify the etiology of otitis media has resulted in an increased recognition of its presence in middle ear fluid. Our finding that A. otitidis is the most prevalent organism in non-purulent effusions is consistent with what has been seen in other recent literature[14, 27, 28].
As was seen in our purulent effusions, S. pneumoniae was also the least identified organism in the non-purulent effusion samples. The overall low prevalence of S. pneumoniae in middle ear fluid in our study is consistent with what is being reported in current OM literature [14, 25, 26, 28–30]. Taken together, our results and current literature may suggest that the pneumococcal conjugate vaccines have significantly reduced the ability of S. pneumoniae to cause recurrent OM.
Another interesting finding lies in the different frequency of identifying each organism when purulent effusions are compared with non-purulent effusions. H. influenzae, S. pneumoniae, and M. catarrhalis were detected ≥2.2x more frequently in purulent effusions than non-purulent effusions. Because OME can persist for several months after AOM[25], decreased frequency of identification of these pathogens from non-purulent effusions may be partly explained by bacterial clearance following antimicrobial treatment and/or clearance by human immune defense mechanisms.
Although the above differences were apparent for the 3 most common otopathogens, A. otitidis results follow a distinct pattern. The fact that A. otitidis percentages remained stable in cases of purulent and non-purulent effusions may suggest that the organism is a commensal. Another possible explanation is that A. otitidis is able to more effectively persist in the middle ear than the three classic otopathogens. According to the literature, strains of A. otitidis lack β-lactamase and can exhibit intermediate levels of resistance to β-lactam antimicrobials as well as cephalosporins[31]. Given that current OM treatment guidelines recommend amoxicillin (a β-lactam) as the first drug of choice, the resistance observed in A. otitidis would decrease the efficacy of this drug. Given that A. otitidis contains no β-lactamase, a combination of amoxicillin and clavulanate would also be less effective against this organism than the classic otopathogens. One could speculate that the described resistances to common OM antimicrobial treatments could facilitate middle ear persistence.
Conclusions
The work presented here describes the prevalence of 4 bacterial otopathogens in middle ear fluid obtained from children undergoing tympanostomy tube placement. Our results suggest that H. influenzae has surpassed S. pneumoniae as the most common pathogen identified in cases of purulent effusions, a result that may have possible implications in antimicrobial treatment guidelines. Although S. pneumoniae was infrequently identified, the odds of a purulent versus non-purulent infection were significantly associated with identification of H. influenzae, S. pneumoniae, and with age <1 year.
Acknowledgments
We would like to thank all the patients and their families who participated in this study. We would also like to thank the medical personnel and staff at Wake Forest Baptist Medical Center who facilitated the completion of this work. We would like to thank Mrs. Tina Swords for her contributions to the project. We would also like to thank the anonymous reviewers whose comments and suggestions have significantly enhanced this manuscript.
Author Contributions
Conceived and designed the experiments: RCH DJK AKE ASW TRP KAP WES SDR. Performed the experiments: RCH DJK AKE ASW SDR. Analyzed the data: RCH DJK AKE ASW TRP KAP WES SDR. Contributed reagents/materials/analysis tools: TRP KAP WES SDR. Wrote the paper: RCH DJK AKE ASW TRP KAP WES SDR.
References
- 1. Schappert SM. Office visits for otitis media: United States, 1975–90. Advance data. 1992;(214):1–19. Epub 1992/08/13. pmid:10126841.
- 2. Klein JO. The burden of otitis media. Vaccine. 2000;19 Suppl 1:S2–8. Epub 2001/02/13. pmid:11163456.
- 3. Finkelstein JA, Davis RL, Dowell SF, Metlay JP, Soumerai SB, Rifas-Shiman SL, et al. Reducing antibiotic use in children: a randomized trial in 12 practices. Pediatrics. 2001;108(1):1–7. pmid:11433046.
- 4. Grijalva CG, Nuorti JP, Griffin MR. Antibiotic prescription rates for acute respiratory tract infections in US ambulatory settings. JAMA: the journal of the American Medical Association. 2009;302(7):758–66. Epub 2009/08/20. pmid:19690308.
- 5. McCaig LF, Besser RE, Hughes JM. Trends in antimicrobial prescribing rates for children and adolescents. JAMA: the journal of the American Medical Association. 2002;287(23):3096–102. Epub 2002/06/19. pmid:12069672.
- 6. Teele DW, Klein JO, Rosner B. Epidemiology of otitis media during the first seven years of life in children in greater Boston: a prospective, cohort study. J Infect Dis. 1989;160(1):83–94. Epub 1989/07/01. pmid:2732519.
- 7. Vergison A, Dagan R, Arguedas A, Bonhoeffer J, Cohen R, Dhooge I, et al. Otitis media and its consequences: beyond the earache. The Lancet Infectious diseases. 2010;10(3):195–203. Epub 2010/02/27. pmid:20185098.
- 8.
Casselbrant ML, Mandel EM. Epidemiology. In: Rosenfeld R. BCD M., editor. Evidence-based Otitis Media: Hamilton: BC Decker Inc.; 2003. p. 147–62.
- 9.
Schwartz SR, Gates GA. Economic costs. In: Rosenfeld RM BC, editor. Evidence-based Otitis Media. 2nd ed: Hamilton: BC Decker Inc.; 2003. p. 333–41.
- 10. Bondy J, Berman S, Glazner J, Lezotte D. Direct expenditures related to otitis media diagnoses: extrapolations from a pediatric medicaid cohort. Pediatrics. 2000;105(6):E72. pmid:10835085.
- 11. Ahmed S, Shapiro NL, Bhattacharyya N. Incremental health care utilization and costs for acute otitis media in children. The Laryngoscope. 2014;124(1):301–5. Epub 2013/05/08. pmid:23649905.
- 12. Alsarraf R, Jung CJ, Perkins J, Crowley C, Alsarraf NW, Gates GA. Measuring the indirect and direct costs of acute otitis media. Archives of otolaryngology—head & neck surgery. 1999;125(1):12–8. Epub 1999/02/05. pmid:9932581.
- 13. Rovers MM. The burden of otitis media. Vaccine. 2008;26 Suppl 7:G2–4. pmid:19094933.
- 14. Holder RC, Kirse DJ, Evans AK, Peters TR, Poehling KA, Swords WE, et al. One third of middle ear effusions from children undergoing tympanostomy tube placement had multiple bacterial pathogens. BMC Pediatr. 2012;12:87. Epub 2012/06/30. pmid:22741759; PubMed Central PMCID: PMC3475091.
- 15. Hendolin PH, Markkanen A, Ylikoski J, Wahlfors JJ. Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions. Journal of clinical microbiology. 1997;35(11):2854–8. pmid:9350746.
- 16.
Bluestone CD, Klein JO. Microbiology. Otitis Media in Infants and Children. Hamilton, ON: BC Decker; 2007. p. 101–26.
- 17. Del Beccaro MA, Mendelman PM, Inglis AF, Richardson MA, Duncan NO, Clausen CR, et al. Bacteriology of acute otitis media: a new perspective. The Journal of pediatrics. 1992;120(1):81–4. Epub 1992/01/01. pmid:1731029.
- 18. Kilpi T, Herva E, Kaijalainen T, Syrjanen R, Takala AK. Bacteriology of acute otitis media in a cohort of Finnish children followed for the first two years of life. The Pediatric infectious disease journal. 2001;20(7):654–62. Epub 2001/07/24. pmid:11465836.
- 19. Long SS, Henretig FM, Teter MJ, McGowan KL. Nasopharyngeal flora and acute otitis media. Infect Immun. 1983;41(3):987–91. Epub 1983/09/01. pmid:6604029; PubMed Central PMCID: PMC264598.
- 20. Block SL, Hedrick J, Harrison CJ, Tyler R, Smith A, Findlay R, et al. Community-wide vaccination with the heptavalent pneumococcal conjugate significantly alters the microbiology of acute otitis media. The Pediatric infectious disease journal. 2004;23(9):829–33. pmid:15361721.
- 21. Casey JR, Pichichero ME. Changes in frequency and pathogens causing acute otitis media in 1995–2003. The Pediatric infectious disease journal. 2004;23(9):824–8. pmid:15361720.
- 22. Casey JR, Adlowitz DG, Pichichero ME. New patterns in the otopathogens causing acute otitis media six to eight years after introduction of pneumococcal conjugate vaccine. The Pediatric infectious disease journal. 2010;29(4):304–9. pmid:19935445.
- 23. McEllistrem MC, Adams J, Mason EO, Wald ER. Epidemiology of acute otitis media caused by Streptococcus pneumoniae before and after licensure of the 7-valent pneumococcal protein conjugate vaccine. J Infect Dis. 2003;188(11):1679–84. Epub 2003/11/26. pmid:14639539.
- 24. McEllistrem MC, Adams JM, Patel K, Mendelsohn AB, Kaplan SL, Bradley JS, et al. Acute otitis media due to penicillin-nonsusceptible Streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2005;40(12):1738–44. Epub 2005/05/24. pmid:15909260.
- 25. Pichichero ME. Otitis media. Pediatric clinics of North America. 2013;60(2):391–407. Epub 2013/03/14. pmid:23481107.
- 26. Zhao AS, Boyle S, Butrymowicz A, Engle RD, Roberts JM, Mouzakes J. Impact of 13-valent pneumococcal conjugate vaccine on otitis media bacteriology. International journal of pediatric otorhinolaryngology. 2014. Epub 2014/01/28. pmid:24461461.
- 27. Harimaya A, Takada R, Hendolin PH, Fujii N, Ylikoski J, Himi T. High incidence of Alloiococcus otitidis in children with otitis media, despite treatment with antibiotics. Journal of clinical microbiology. 2006;44(3):946–9. pmid:16517881.
- 28. Khoramrooz SS, Mirsalehian A, Emaneini M, Jabalameli F, Aligholi M, Saedi B, et al. Frequency of Alloicoccus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in children with otitis media with effusion (OME) in Iranian patients. Auris, nasus, larynx. 2012;39(4):369–73. Epub 2011/08/27. pmid:21868180.
- 29. Vijayasekaran S, Coates H, Thornton RB, Wiertsema SP, Kirkham LA, Jamieson SE, et al. New findings in the pathogenesis of otitis media. The Laryngoscope. 2012;122 Suppl 4:S61–2. Epub 2013/01/04. pmid:23254605.
- 30. Kim SJ, Chung JH, Kang HM, Yeo SG. Clinical bacteriology of recurrent otitis media with effusion. Acta oto-laryngologica. 2013;133(11):1133–41. Epub 2013/10/16. pmid:24125184.
- 31. Bosley GS, Whitney AM, Pruckler JM, Moss CW, Daneshvar M, Sih T, et al. Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media. Journal of clinical microbiology. 1995;33(11):2876–80. Epub 1995/11/01. pmid:8576338; PubMed Central PMCID: PMC228599.