This prospective, epidemiological study was conducted at seven centres across Spain between February 2009 and May 2012 (GSK study identifier: 111425). Children aged ≥3 to 36 months with ear–nose–throat specialist-confirmed AOM, and who could provide a middle ear fluid sample (MEF), were enrolled in the study. MEF samples were collected by tympanocentesis; in case of otorrhea, the samples were collected via deep aspiration of MEF through needle insertion after ear canal cleaning.

AOM was confirmed based on the acute and sudden onset of the disease within the previous 3 days: fever, irritability, intense erythema of the tympanum or earache; and presence of effusion in the middle ear. The presence of middle ear effusion was characterized by tympanum bulging, limited or no mobility in the tympanum (evaluated using pneumatic otoscopy), fluid behind the tympanum and/or otorrhea (defined as perforation occurring within 48h before enrollment).

Children were excluded from participating in the study if they were hospitalized during the AOM episode (to exclude possible confusion with intrahospital infections) or in the event of otitis externa/otitis media with effusion: AOM for >72h or otorrhea for >48h before enrollment; or in situ transtympanic aerators. Children who received antibiotics for illnesses other than AOM during the 72-hour period before enrollment and were prescribed antibiotics before performing tympanocentesis were also excluded. Finally, patients with protocol-forbidden medical conditions were excluded.

The study adhered to the principles of Good Clinical Practice, including the 1964 Declaration of Helsinki and local Spanish rules and regulations. The study was approved by the Institutional Review Board of each participating center and parents/guardians provided written informed consent before enrollment.

MEF samples were collected by tympanocentesis performed under otoscopy through a reusable CDT Aspirator attached to a CDT speculum, or by sampling of spontaneous otorrhea. In cases of bilateral AOM, MEF samples were collected from both ears. Tympanocentesis was performed without anesthesia. Aspirated MEF samples were inoculated onto Amies transport medium11 and kept at room temperature for 16h (and up to a maximum of 48h) before subsequent analysis at a GSK-designated laboratory. MEF samples were then inoculated onto chocolate (with bacitracin for otorrhea samples) and blood agar (with nalidixic acid). Bacterial identification was undertaken using standard bacteriological procedures.12 Serotyping for Spn was performed by Quellung's reaction and for Hi using monovalent anti-sera.

Culture-negative samples were further analyzed using real-time PCR assay for the presence of the pneumococcal pneumolysin (ply) gene,13 pneumococcal wzg gene,14 and the omp2 gene for non-capsulated and capsulated Hi.15 Inhibited PCR reactions were all checked using a commercial exogenous internal positive control (Applied Biosystems, Foster City, CA, USA). A subsequent extraction for eliminating inhibitors was performed on inhibited samples using 20% (w/v) Chelex-100 resin (BioRad Laboratories, Hercules, CA, USA). The samples were further tested for antibacterial susceptibility. Minimal inhibitory concentrations were derived using Etest (bioMérieux, France) and interpreted with the criteria published by the Clinical and Laboratory Standards Institute in 2009.16

Analyses were performed on children meeting all the selection criteria, complying with protocol-defined procedures, and from whom laboratory results of the MEF sample were available. The proportion of AOM episodes caused by Spn, Hi and other bacterial pathogens and the percentages of Spn and Hi serotypes were calculated. Only the first AOM episode was included and any recurrent AOM cases were excluded from analyses. The antibacterial susceptibility of Spn and Hi was determined. History of previous PCV, Hi-type B and influenza vaccinations was recorded. All statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA).

Of 125 AOM episodes in 124 subjects, recorded during the study period, 117 episodes (tympanocentesis=91; otorrhea=26) (from 117 subjects) were analyzed. Only one subject had two AOM episodes, of which one episode was eliminated from the analyses. Therefore, a total of eight AOM episodes were excluded from the analyses: protocol violation (n=1), having a protocol-forbidden medical condition (n=4) and missing MEF/otorrhea sample (n=3). The median age of children was 17 months (range: 3–35) and 60.7% were male. AOM episodes were most frequently observed in children aged 11–22 months (46.2%; 54/117) followed by 23–35 months (32.5%; 38/117) and 3–10 months (21.4%; 25/117). At least one dose of a pneumococcal vaccine and Hi-type B vaccine was received by 69.2% and 95.7% subjects, respectively. None of the subjects received influenza vaccine. Table 1 presents information on age groups in the study, summary of vaccination history and antibiotic treatment.

The microbiological results are reported in Table 2. Bacterial culture tests were performed for 117 episodes. Growth for any bacterium was cultured from 66% (77/117) of episodes: 82% of which (63/77) were culture-positive for at least one bacterium under study (Spn, Hi, Streptococcus pyogenes [Spy] or Moraxella catarrhalis [Mcat]). The most frequent bacteria were Hi (42%; 32/77) and Spn (31%; 24/77). Spy was isolated in 10 samples and Mcat in 1 sample; co-infection was detected in 10 episodes, of which 4 co-infections involved bacterium under study: Spn and Hi (n=3); Spy and Hi (n=1). The remaining 40 episodes were culture-negative. However, PCR was performed for 42 episodes (40 culture-negative episodes+2 culture-positive episodes), of which 48% (n=20) were found to be Hi-positive and 55% (n=23) were Spn-positive.

Combined culture and PCR results indicated that 52 AOM episodes (44%) were Hi-positive and 46 (39%) were Spn-positive; 17 episodes (15%) were positive for both Hi and Spn.

The most common Spn serotypes amongst culture-positive episodes were 19F (17%; 4/24) and 19A (13%; 3/24) (Fig. 1). In episodes from children who had been previously vaccinated, PCV7 vaccine serotypes (14 and 19F) were present in 19% (3/16) of episodes and PCV7/PCV13 vaccine serotype (6B) was present in 6% (1/16) of episodes (Table 3). PCV7 vaccine serotype (19F) was present in 25% (2/8) episodes from unvaccinated children.

Figure 1.

Serogroup and serotype distribution of Streptococcus pneumoniae-positive episodes (N=24). n=number of Streptococcus pneumoniae-positive episodes with respective serotypes and serogroups. OTHER=serogroups 8, 15, 22, 35 (1 episode each) and 21 (2 episodes).

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All 32 Hi-positive episodes were non-typeable (NTHi): 69% (n=22) in vaccinated (PCV vaccine) and 31% (n=10) in unvaccinated (PCV vaccine) children.

Of the 24 Spn-positive episodes, all (100%) were susceptible to cefotaxime, 22 (92%) to amoxicillin, 17 (71%) to erythromycin and 17 (74%) to cefpodoxime (since there was one missing Spn-positive episode susceptible to only cefpodoxime, 23 of 24 Spn-positive episodes were considered for the analyses). Ten episodes were susceptible and intermediately susceptible to penicillin G and four (17%) were resistant to penicillin (Fig. 2). Serotype 19A was most commonly resistant to penicillin G, amoxicillin, cefpodoxime, and erythromycin.

Figure 2.

Antibacterial susceptibility of Streptococcus pneumoniae-positive episodes (N=24).

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Of the 32 Hi-positive episodes, all (100%) were susceptible to both cefpodoxime and cefotaxime and 26 (81%) to amoxicillin/clavulanic acid. Six episodes (19%) were resistant to amoxicillin/clavulanic acid (Fig. 3).

Figure 3.

Antibacterial susceptibility of Haemophilus influenzae-positive episodes (N=32).

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Multi-drug resistance was observed in 17% (4/24) Spn-positive episodes: one episode was resistant to penicillin G, cefpodoxime and erythromycin; and three episodes were resistant to penicillin G, amoxicillin, cefpodoxime and erythromycin. Multi-drug resistance was observed in six NTHi-positive episodes (19%).

Of the 81 children positive for at least one pathogenic bacteria by combined culture+PCR, 55 (68%) children had received the appropriate PCV doses according to their age: 61% (11/18) aged 3–10 months, 64% (21/33) aged 11–22 months and 77% (23/30) aged 23–35 months. The vaccines administered corresponded to PCV7 in 62 children (77%, where 44 children [71%] received ≥3 doses), PCV10 in nine (11%, where six children [67%] received ≥3 doses) and PCV13 in 17 children (21%, where two children [12%] received ≥3 doses). There was no statistically significant difference in terms of vaccination between Hi-positive (P=1.000) and Spn-positive children (P=0.9497).