Eur J Pediatr (2015) 174:355–364 DOI 10.1007/s00431-014-2409-0

ORIGINAL ARTICLE

Bacterial spectrum of spontaneously ruptured otitis media in the era of pneumococcal conjugate vaccination in Germany Mark van der Linden & Matthias Imöhl & Andreas Busse & Markus Rose & Dieter Adam

Received: 17 June 2014 / Revised: 30 July 2014 / Accepted: 19 August 2014 / Published online: 30 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Otitis media is a common pediatric disease and the main reason for antibiotic prescription in children. Before implementation of routine childhood pneumococcal vaccination in Germany, serotypes contained in the seven-valent Communicated by Peter de Winter Author’s summary This study examines the bacteriology of middle ear fluids and nasopharyngeal carriage of children with spontaneously perforated AOM, thus providing surveillance data on pneumococcal disease and carriage in the era of routine pneumococcal vaccination in Germany. Following universal PCV7 immunization, a clear epidemiological impact of pneumococcal conjugate vaccination was observed, as PCV7 serotypes have almost disappeared among AOM. Among the remaining serotypes, 3 and 19A are the most prevalent, indicating the added value of higher-valent pneumococcal conjugate vaccination. Concerning carriage, the percentage of children carrying pneumococci has not changed. However, the serotype distribution of carriage has strongly shifted toward non-PCV7 serotypes, showing that vaccination has the ability to replace serotypes common in AOM with ones that are probably less harmful. This might particularly be the case after the effects of PCV13 vaccination on AOM become visible in the next study years. Mark van der Linden and Matthias Imöhl contributed equally to this study. M. van der Linden : M. Imöhl National Reference Center for Streptococci, Department of Medical Microbiology, University Hospital (RWTH), Aachen, Germany A. Busse Pediatric Medical Practice, Tegernsee, Germany M. Rose Children’s Hospital, Goethe University, Frankfurt, Germany D. Adam Children’s Hospital, University of Munich, Munich, Germany M. van der Linden (*) Department for Medical Microbiology, National Reference Center for Streptococci, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany e-mail: [email protected]

pneumococcal conjugate vaccine (PCV) were among the most frequent pneumococcal serotypes responsible for acute otitis media (AOM). This report describes the first 3 years of a prospective, multicenter, epidemiological cross-sectional study examining the bacteriology of middle ear fluids (MEF) and nasopharyngeal swabs (NPS) of children 2 months to 5 years of age with spontaneously perforated AOM in the era of routine pneumococcal vaccination. MEF was obtained from 963 subjects; NPS from 877. Reported case numbers steeply decreased over the three study years even though the recruiting base remained the same. Among subjects with relevant bacterial growth in their MEF swabs, 113 (11.7 %) had Streptococcus pyogenes, 97 (10.1 %) Staphylococcus aureus, 88 (9.1 %) Streptococcus pneumoniae, 63 (6.5 %) Haemophilus influenzae, and 8 (0.8 %) Moraxella catarrhalis. S. pneumoniae isolates decreased from 41 (9.3 %) in year 1 to 12 (5.7 %) in year 3 (p=0.128). PCV7 serotypes accounted for only 7.9 % (n=7) of isolated pneumococci. Of the 877 subjects with NPS cultures, 465 (53.0 %) carried S. pneumoniae, 314 (35.8 %) H. influenzae, 292 (33.3 %) M. catarrhalis, and 110 (12.5 %) S. pyogenes; 79.4 % (n=765) of the children were vaccinated with at least one dose of PCV. Carriage of pneumococci was slightly lower in vaccinated (47.8 %) than in unvaccinated (52.7 %) children (p=0.254). PCV7 serotypes were carried by 9.6 % of unvaccinated children but by only 4.2 % of vaccinated children, resulting in a 56.3 % vaccine effectiveness. Conclusions: Following universal PCV7 immunization, a clear epidemiological impact of pneumococcal conjugate vaccination was observed as PCV7 serotypes have almost disappeared among AOM. Keywords Otitis media . Vaccination . Germany Abbreviations AOM

Acute otitis media

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GAS IPD MEF NIP NIPD NPS NT-H. influenzae PCV PNSP

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Group A Streptococcus Invasive pneumococcal disease Middle ear fluid National immunization program Noninvasive pneumococcal disease Nasopharyngeal swab Nontypeable Haemophilus influenzae Pneumococcal conjugate vaccine Penicillin nonsusceptible Streptococcus pneumoniae

Introduction Acute otitis media (AOM) is the number one reason for pediatric consultations and antibiotic prescriptions in industrialized countries [4]. Streptococcus pneumoniae infections are one of the leading causes of morbidity and mortality among infants and children worldwide [26]. S. pneumoniae may cause invasive pneumococcal disease (IPD), such as bacteremic pneumonia and meningitis, as well as noninvasive pneumococcal disease (NIPD) like otitis media, sinusitis, bronchitis, and nonbacteremic pneumonia. Irrespective of being less severe, NIPDs have a great impact on public health: nearly every child has experienced an episode of AOM by the age of 2 years [20]. AOM is a painful inflammation of the middle ear with a multifactorial pathogenesis. It is usually based on an ascending, either existent or precedent, infection of the upper respiratory system via the Eustachian tube. Due to swelling of the tube, drainage is reduced and the tympanum might perforate spontaneously [3]. Results of previous AOM studies consistently revealed S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes as major bacterial pathogens in the development of AOM [7, 31]. AOM is the main reason for antibiotic prescriptions in children, promoting antibiotic resistance not only in pneumococci, but also in other pathogens [15, 34]. Therefore, prevention of pneumococcal infections through vaccines is of utmost importance [8, 11]. In Germany, routine childhood pneumococcal vaccination started in 2006. Until the end of 2009, a heptavalent proteinconjugated CRM197-vaccine (PCV7) containing five of the most frequent pneumococcal serotypes (i.e., 6B, 9V, 14, 19F, and 23F [6, 10]) responsible for AOM was predominantly used for routine vaccination in young children. Strains with these serotypes also make up most of the penicillinnonsusceptible S. pneumoniae strains (PNSP) [6, 23, 33]. Several clinical trials in other countries demonstrated a significant reduction of AOM in PCV7-immunized children. Furthermore, the overall incidence of pneumococcal disease

was reduced in PCV7-immunized children, as well as the occurrence of vaccine-serotype-associated infections. By contrast, nonvaccine-serotype-associated infections were slightly elevated [5, 14, 17, 19]. Worldwide, an increase of nonvaccine-serotype pneumococci has been observed. This phenomenon was first reported in countries which have implemented PCV7 national immunization programmes (NIP) [25]. Remarkably, increases of nonvaccine-serotypes were also observed in the absence of pneumococcal NIPs and might thus be due to fluctuation (e.g., Korea [12] and Israel [15]), which has been observed in the decades before the availability of PCVs as well [18, 22]. In 2006, a general recommendation for childhood pneumococcal vaccination was issued in Germany [1] with the potential not only to be effective against IPD but also to have an impact on AOM and bacterial colonization. According to WHO recommendations, vaccination programmes should be accompanied by surveillance of the addressed bacteria. With the availability of higher-valent pneumococcal conjugate vaccines (PCV10 with additional serotypes 1, 5, and 7F and PCV13 with additional serotypes 1, 3, 5, 6A, 7F, and 19A, both licensed in 2009 [2]), data of serotype distributions among children with AOM can be used to estimate the potential impact of these broader vaccines. This study examines the bacteriology of middle ear fluids (MEF) and nasopharyngeal carriage of children with spontaneously perforated AOM, thus providing surveillance data on pneumococcal disease and carriage in the era of routine pneumococcal vaccination in Germany.

Materials and methods Study design This prospective, multicenter, epidemiological cross-sectional study included children 2 months to 5 years of age with acute, spontaneously draining otitis media. Since aspiration of MEF for study purposes is considered to be unethical in Germany, only cases of spontaneously draining AOM could be included. Nasopharyngeal swabs (NPS) were taken when possible. The immunization status of the subjects was documented. Our analysis encompassed: (1) description of the study population (demographic data, environmental influences, relevant concomitant diseases, and immunization status), (2) analysis of frequencies and distribution of bacterial strains in the MEF and in the NPS samples, (3) analysis of the serotype/ emm-type distribution of S. pneumoniae, H. influenzae, and S. pyogenes found in the ear swab samples and in the NPS samples, and (4) intra-individual correlation between the S. pneumoniae, H. influenzae, and S. pyogenes serotype/ emm-type distribution in the MEF and the NPS samples.

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Sample size determination was based on the assumption that approximately 10 % of the MEF samples from subjects with AOM will be positive for S. pneumoniae. Since approximately six to eight cases of spontaneously draining AOM are seen per year in a pediatrician’s office, 50 sites were considered to be necessary in order to gain 1,000 samples of MEF from subjects with a spontaneous draining AOM within 36 months. This interim report is based on the evaluation of all subjects included during the first (15 Oct 2008 to 14 Oct 2009), second (15 Oct 2009 to 14 Oct 2010), and third (15 Oct 2010 to 14 Oct 2011) year of the study. The primary objective of the study was the description of the bacterial microbiology findings of children with an acute, spontaneously draining otitis media. Subjects were identified in the offices of 50 initiated study sites of practicing pediatricians in Germany, out of which 43 sites have been actively recruiting during the first three study years. In September 2011, 35 more sites were initiated, of which nine had already started recruiting within the last month of the third study year, resulting in 24 recruited subjects. At the first visit, demographic data including environmental parameters, relevant antecedent diseases, and immunization status were documented in a questionnaire. The pediatrician obtained MEF by ear swab sampling. If possible (depending on the child’s condition and parental consent), an additional NPS sample was taken as well. The development and status of the AOM was supposed to be evaluated at max 14 days after the inclusion visit, at visit 2. According to the clinical routine, tympanometry or audiometry was performed (depending on the child’s age) to confirm that the AOM had resolved. If the AOM was not resolved at visit 2, a follow-up visit (approximately 30 days after the inclusion visit) was scheduled. To exclude seasonal influences, the study was performed throughout the year. For all participants, written informed consent was obtained prior to the study. The human experimentation guidelines of Good Clinical Practice, the German Drug Act and the Declaration of Helsinki/Hong Kong were followed. Approval was obtained from the local ethical committee.

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Bacterial growth conditions Primary material (MEF and NPS) was plated on the following media: Columbia blood agar, Mc Conkey agar, chocolate agar, and Sabouraud agar. Identification was obtained through routine microbiology techniques and MALDI-TOF (Bruker, Germany).

Serotyping Serotyping of S. pneumoniae-positive isolates was performed according to the “Neufeld Quellung” reaction, with type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark. H. influenzae was serotyped using Difco H. influenzae antisera from Becton Dickinson, USA. The emm-typing of S. pyogenes was performed as follows: PCR for amplification of the emm gene was carried out using the “all M” primers described by Podbielski et al. [27]. PCR products were purified and sequenced using an automated ABI Prism 310 DNA sequencer (Applied Biosystems, Weiterstadt, Germany). The nucleotide sequences encoding the N-terminal hypervariable portion of the M protein were compared with the sequences published on the Website of the Centers for Disease Control and Prevention (http://www.cdc. gov/ncidod/biotech/strep/strepblast.htm). In case of a high degree of identity (95 % or more) with the 5′-terminal portion of a known sequence, the nucleotide sequence was given the corresponding type designation.

Vaccine effectiveness and statistical calculations Vaccine effectiveness was calculated using the indirect cohort method as described by Broome et al. [9]. To compare differences in carriage and changes in MEF, Fisher’s exact test was calculated using Microsoft Excel 2010 and two-sided p values were reported.

Selection of study population Results Each subject was included only once in this study. Inclusion criteria were: age 2 months to 5 years, active, and spontaneously draining AOM for a maximum of 2 days. Exclusion criteria were: previous ear surgery, chronic otitis media with effusion for at least 3 days, any severe chronic diseases and/or immunodeficient conditions, use of systemic steroids or other immunosuppressants, craniofacial malformations, treatment with antibiotics over a timeframe of more than 48 h prior to inclusion in this study, relevant hemorrhagic diseases, and participation in other interventional studies or clinical trials.

In the first 3 years of the study, a total of 994 subjects were recruited. Of those, 963 were included, and MEF samples were obtained. A total of 31 subjects were excluded after application of the inclusion/exclusion criteria. For 877 subjects, an NPS isolate was obtained; for 86 subjects, an NPS isolate could not be obtained (Table 1). In the first study year, 443 subjects were included; in the second year, 310; and in the third year, 210, even though the recruiting base remained the same (Fig. 1).

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Table 1 Numbers of subjects included in the study Subjects

Year 1

Included Excluded Total

Year 2

Year3

All

MEF

NPS

MEF

NPS

MEF

NPS

MEF

NPS

443 24 467

391 76 467

310 4 314

294 20 314

210 3 213

192 21 213

963 31 994

877 117 994

Reasons for exclusion: age (n=12), previous ear operation (n=5), chronic disease (n=1), and technical errors (n=13)

Demographics The age of the subjects varied between 2 and 71 months. The average age was 29.6 months (median of 28 months). Male subjects slightly outnumbered females (511, 53.1 %); 295 (30.6 %) children had no siblings, 442 (45.9 %) had one sibling, and 226 (23.5 %) had 2 or more siblings. Of the children, 528 (54.8 %) attended daycare and 183 (19.0 %) were regularly exposed to cigarette smoke.

MEF isolates Of the 963 obtained MEF samples, 144 (15 %) showed no bacterial growth and 478 (49.6 %) showed physiological outer-ear flora or temporary contamination of the auditory canal, defined as bacterial strains other than S. pyogenes, S. aureus, S. pneumonia, H. influenzae, and M. catarrhalis. Relevant bacteria were isolated from 341 subjects (35.4 %). Of these 341, 313 subjects (91.8 %) had a single bacterial strain isolated; for 27 (7.9 %), 2 isolates were obtained, and 1 subject (0.3 %) had 3 isolates.

500

450 400 350 300 250 200 150 100

50 0 year 1

year 2

year 3

Fig. 1 Decreasing case numbers at constant recruiting basis. Black bars, MEF isolates; gray bars, NPS isolates

Among the 341 patients with relevant bacterial growth in their MEF swabs, S. pyogenes was recovered in 113 (11.7 %), S. aureus in 97 (10.1 %), S. pneumoniae in 88 (9.1 %), H. influenzae in 63 (6.5 %), and M. catarrhalis in 8 (0.8 %) cases (Table 2). For a total of 27 patients, two relevant strains were isolated (S. pyogenes/S. aureus, n=9; S. pneumoniae/ S. aureus, n = 8; S. pneumoniae/H. influenzae, n = 5; H. influenzae/S. aureus, n=2; and M. catarrhalis/S. aureus, S. pneumoniae/S. pyogenes, and S. pneumoniae/ S. pneumoniae, n=1 each). In one patient, three relevant strains were detected (S. pneumoniae/H. influenzae/ S. aureus). For a total of 133 patients, other bacteria or molds (Bacillus, Pseudomonas, Enterococcus, Acinetobacter, Enterobacter, Klebsiella, Pantoea, Escherichia coli, C o r y n e b a c t e r i u m , S e r r a t i a , S t e n o t ro p h o m o n a s , Propionibacterium, Lactobacillus, Comamonas, Citrobacter, Alcaligenes, and Candida) were found, which were not further analyzed. Nasopharyngeal swabisolates An NPS isolate was obtained for 877 subjects. The isolates were analyzed for the presence of the following bacteria: S. pneumoniae, S. pyogenes, H. influenzae, and M. catarrhalis. Of the 877 patients, 465 (53.0 %) carried S. pneumoniae, 314 (35.8 %) H. influenzae, 292 (33.3 %) M. catarrhalis, 110 (12.5 %) S. pyogenes, and 142 (16.2 %) carried none of the above strains. Percentages do not add up to 100, because multiple carriage was observed (Table 2). S. pneumoniae The percentage of patients with S. pneumoniae isolated from MEF was 9.3 % (41 of 443 patients) in year 1 and 11.3 % (35 of 310) in year 2, but decreased in year 3 (5.7 %; 12 of 210). However, the amount of S. pneumoniae isolated decreased in conjunction with lower numbers of AOM cases reported and for almost every serotype (Fig. 2). The most prevalent serotypes in all three study years were serotypes 3 and 19A. Serotypes included in PCV7 accounted for 7.1, 8.6, and 8.3 % of isolated pneumococci in study years 1, 2, and 3, respectively, and PCV10 serotypes for 11.9, 20.0, and 16.7 %. Serotypes included in PCV13 accounted for 69.0 % (year 1), 68.6 % (year 2), and 83.3 % (year 3), with very low case numbers in the third study year (Table 3). The percentage of subjects with S. pneumoniae-positive NPS was 49.9 % in year 1, 56.5 % in year 2, and 54.2 % in year 3 (Table 2). The serotype distribution of these isolates did not change dramatically over the three study years. PCV7 serotypes were found in 14.2 % of the isolates in year 1 (year 2, 9.4 %; year 3, 9.9 %; p=0.294). PCV10 serotypes made up for 17.6 % of the isolated pneumococci in year 1 (year 2, 14.6 %; year 3, 11.7 %; p = 0.194). PCV13 serotypes

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Table 2 Bacterial isolates from MEF and NPS of 963 subjects with severe AOM Year 1

MEF isolates Streptococcus pneumoniae Haemophilus influenzae Moraxella catarrhalis Streptococcus pyogenes Staphylococcus aureus Patients with 1 of above Patients with 2 of above Patients with 3 of above Patients with none of above Total NPS isolates S. pneumoniae H. influenzae M. catarrhalis S. pyogenes Patients with 1 of above Patients with 2 of above Patients with 3 of above Patients with 4 of above Patients with none of above Total

Year 2

Year 3

n

Patientsa

%b

n

Patientsa

%b

42 32 7 54 46 153 28 0 0 181

41 32 7 54 46 153 14 0 276 443

9.3 7.2 1.6 12.2 10.4 34.5 3.2 0.0 62.3 100.0

35 17 1 34 34 98 20 3 0 121

35 17 1 34 34 98 10 1 201 310

11.3 5.5 0.3 11.0 11.0 31.6 3.2 0.3 64.8 100.0

204 142

195 142

49.9 36.3

171 109

166 108

115 54 181 228 102 4 0 515

115 54 181 114 34 1 61 391

29.4 13.8 46.3 29.2 8.7 0.3 15.6 100.0

97 39 116 188 108 4 0 416

97 39 116 94 36 1 47 294

Total Patientsa

%b

12 14 0 25 17 62 6 0 0 68

12 14 0 25 17 62 3 0 145 210

5.7 6.7 0.0 11.9 8.1 29.5 1.4 0.0 69.0 100.0

56.5 36.7

111 66

104 64

33.0 13.3 39.5 32.0 12.2 0.3 16.0 100.0

82 18 69 126 66 16 0 277

80 17 69 63 22 4 34 192

n

Patientsa

%b

89 63 8 113 98 313 54 3 0 370

88 63 8 113 97 313 27 1 622 963

9.1 6.5 0.8 11.7 10.1 32.5 2.8 0.1 64.6 100.0

54.2 33.3

486 317

465 314

53.0 35.8

41.7 8.9 35.9 32.8 11.5 2.1 17.7 100.0

294 111 366 542 276 24 0 1208

292 110 366 271 92 6 142 877

33.3 12.5 41.7 30.9 10.5 0.7 16.3 100.0

n

a

For one patient two different S. pneumoniae MEF isolates were found. Among NPS isolates multiple carriage of the same genus was found on multiple occasions

b

Percentage of all included patients for whom MEF was obtained

Fig. 2 Amount of S. pneumoniae isolates from MEF, per serotype and per study year

16 3 14

19A 19F

Number of reported cases

12

1 7F

10

others

8 6 4 2 0 year 1

year 2

Children vaccinated with PCV7/PCV10/PCV13/mixed (n=, %): year1: 312, 98.1 / 1, 0.3 / 1, 0.3 / 4, 1.3 year2: 206, 84.1 / 11, 4.5 / 12, 4.9 / 16, 6.5 year3: 132, 65.3 / 6, 3.0 / 43, 21.3 / 21, 10.4

year 3

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Table 3 Serotype distribution in Streptococcus pneumoniae isolates from MEF and NPS Serotype MEF PCV7 19F PCV10 1 7F PCV13 3 19A Non-PCV13 Total NPS PCV7 4 6B 9V 14 18C 19F 23F PCV10 1 5 7F PCV13 3 6A 19A Non-PCV13 6C 22F 23B 35B NT Others Total

Year 1

Percent

Year 2

Percent

Year 3

Percent

Percent*

3 3 5 2 0 29 15 9 13 42

7.1 7.1 11.9 4.8 0.0 69.0 35.7 21.4 31.0 100.0

3 3 7 2 2 24 9 8 12 35

8.6 8.6 20.0 5.7 5.7 68.6 25.7 22.9 34.3 100.0

1 1 2 0 1 10 4 4 2 12

8.3 8.3 16.7 0.0 8.3 83.3 33.3 33.3 16.7 100.0

29 0 6 1 0

14.2 0.0 2.9 0.5 0.0

16 0 2 2 1

9.4 0.0 1.2 1.2 0.6

11 1 1 0 1

9.9 0.9 0.9 0.0 0.9

1 15 6 36 5 0 2 114 50 7 21 90 2 3 3 0 0 82 204

0.5 7.4 2.9 17.6 2.5 0.0 1.0 55.9 24.5 3.4 10.3 44.1 1.0 1.5 1.5 0.0 0.0 40.2 100.0

1 9 1 25 3 1 5 83 35 5 18 88 7 4 7 5 2 61 171

0.6 5.3 0.6 14.6 1.8 0.6 2.9 48.5 20.5 2.9 10.5 51.5 4.1 2.3 4.1 2.9 1.2 35.7 100.0

0 7 1 13 0 0 2 66 30 5 18 45 2 5 4 4 4 23 111

0.0 6.3 0.9 11.7 0.0 0.0 1.8 59.5 27.0 4.5 16.2 40.5 1.8 4.5 3.6 3.6 3.6 20.7 100.0

Non-PCV13 MEF: 6C (2), 11A (4), 15A (1), 15B/C (2), 21 (3), 22F (1), 23A (3), 23B (4), 31 (1), 33F (1), 35B (1), 35F (1), NT (1), and not typed (1). Other NPS: 9N (3), 10A (9), 11A (31), 15A (4), 15B/C (31), 16F (2), 17A (1), 17F (3), 18A (1), 21 (11), 23A (19), 24A (1), 24F (2), 28A (1), 31 (6), 33F (11), 34 (7), 35C (1), 35F (18), and 38 (8) *Statistical significance for year 1 compared with year 3: PCV7, p=0.294; PCV10, p=0.194; PCV13, p=0.553; 6C, p=0.615; 22F, p=0.135; 23B, p= 0.247; 35B, p=0.015; NT, p=0.015

accounted for 55.9 % in year 1 (year 2, 48.5 %; year 3, 59.5 %; p=0.553). Among the isolates with non-PCV13 serotypes, an increase in serotypes 6C, 22F, 23B, 35B, and nontypeables (NT) was observed but was only significant for 35B and NT (p=0.015 in both cases) (Table 3).

H. influenzae, M. catarrhalis, S. pyogenes, and S. aureus H. influenzae was found in MEF of 7.2 % (n=32) of the subjects in year 1, 5.5 % (n=17) in year 2, and 6.7 % (n= 14) in year 3. Nontypeable H. influenzae (NT-H. influenzae)

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made up for 79.4 % (87.5, 70.6, and 71.4 %) of these isolates (capsulated isolates included serotypes b and f). Among NPS isolates, H. influenzae was found in 36.3, 36.7, and 33.3 % of the subjects in study years 1, 2, and 3, respectively, with 90.8, 77.1, and 71.2 % NTHi (capsulated isolates included serotypes a, b, e, and f). M. catarrhalis was found in less than 2 % of the MEF samples and found in 30–40 % of NPS samples. S. pyogenes was found in about 12 % of MEF samples and about 9–14 % of NPS samples. Most common emm-types were 1 (31.9 %), 12 (11.5 %), 89 (10.6 %), 6 (8.8 %, 28 (8.0 %), 3 (7.1 %), and 75 (6.2 %), and 12 S. aureus was found in 10.4 % of MEF samples in year 1 (year 2, 11.0 %; year 3, 8.1 %). Among NPS samples, a screening for S. aureus was not performed (Table 2). Comparison of MEF and NPS isolates A comparison of isolates from MEF and NPS obtained from the same subject showed that isolates were identical for 96.1 % of S. pneumoniae (judged by serotype, 49 out of 51 MEF-NPS sample pairs), 96.9 % of S. pyogenes (judged by emm-type, 63 out of 65 MEF-NPS sample pairs), and 87.2 % of H. influenzae (judged by serotype, 34 out of 39 MEF-NPS sample pairs). Vaccination status data In our study population, 79.4 % (n=765) of the children had been immunized with at least one dose of PCV (unvaccinated, n=188; unknown, n=10). Of these, 703 (91.9 %) were vaccinated according to age recommendations. Among 61 children not vaccinated according to age, 9 (14.8 %) had received an incomplete primary series and 52 (85.2 %) had received no booster (for one child, information on timeliness of vaccination was not available). Among the 52 children that had received no booster, 32 had an incomplete primary series as well. Most children had been vaccinated with PCV7 (85.1 %). PCV13 accounted for 7.6 % of vaccinations and PCV10 for 2.4 %. Thirty-four (4.4 %) children were vaccinated with

mixed immunization schedules (PCV7/-13, 2.6 %; PCV7/10, 1.0 %; PCV10/-13, 0.5 %; PCV10/-7, 0.1 %; and PCV11/7, 0.1 %). For four children, the vaccine used could not be ascertained. Children who were vaccinated with PCV7 according to age (n=598) had AOM caused by S. pneumoniae in 9.4 % (n=56) of the cases. Only four of them (0.7 %) had a vaccine serotype (19F in all four cases). Among the 188 nonvaccinated children, 18 (9.6 %; p = 0.887) had AOM caused by S. pneumoniae and only one (0.5 %; p=1.000) vaccine type (19F) was found. Carriage of pneumococci among children with AOM was slightly lower in vaccinated (47.8 %) than in unvaccinated (52.7 %) children (p=0.254). Children vaccinated with PCV7 according to age (n=598) carried S. pneumoniae in 281 cases (47.0 %). Only 25 (4.2 %) of the carried isolates showed PCV7 serotypes. Among unvaccinated children (n=188), 91 (48.4 %) carried S. pneumoniae and 18 (9.6 %) of the carried isolates showed a PCV7 serotype. Using the indirect cohort method [9], this resulted in a 56.3 % vaccine effectiveness of PCV7 (at least one dose) on carriage. The postprimary (all patients that had received three doses of PCV7, but no booster) effectiveness for PCV7 was 52.2 %, and the postbooster (all patients that had received a booster dose of PCV7) effectiveness was 69.6 % (Table 4). Numbers for calculating the vaccine effectiveness for AOM were too small. Carriage of H. influenzae (33.7 vs. 24.5 %; p=0.015), M. catarrhalis (31.9 vs. 23.9 %; p=0.034) and S. pyogenes (11.9 vs. 9.6 %; p=0.443) was higher in vaccinated than in unvaccinated children.

Discussion The major strength of this study is that it is the first systematical analysis of the microbiology of the MEF from children with spontaneously draining otitis media in Germany. Results from hitherto existing bacteriological studies on AOM

Table 4 Nasopharyngeal carriage of Streptococcus pneumoniae according to vaccination status All Patients vaccinated with PCV7 according to age Patients not vaccinated Patients vaccinated with 3 primary doses of PCV7 (postprimary) Patients not vaccinated (age ≥5 and