Streptococcus pneumoniae (Sp), Haemophilus influenzae (Hi) and in a lesser extent Moraxella catarrhalis (Mc) are the main bacterial species implicated in acute otitis media (AOM),1–3 the leading reason for antibiotics prescription in childhood.4 , 5 Even if most episodes of AOM resolve spontaneously,6 , 7 in most countries, antibiotics are indicated for AOM in young children to reduce the duration of symptoms and the risk of complications.8 , 9 The choice of antibiotic prescriptions must consider many factors including the bacterial species implicated and their level of resistance to antibiotics but also the tolerability and the potential ecological impact of the different compounds.
The reservoir of bacterial species implicated in AOM is the nasopharynx, and their carriage precedes AOM.10 , 11 Although NP cultures are not useful to individually predict the etiology of AOM,12 , 13 they have been proposed for following the trends of antibiotic susceptibility and the impact of pneumococcal conjugate vaccines (PCVs).14 , 15 The NP carriage of Sp and Hi varies according to several factors such as number of siblings, crowding, day care center (DCC) attendance, respiratory tract infections, and also antibiotics use and vaccine selective pressure.16 Contrary to Sp and Hi, almost all strains of Mc are β-lactamase–producing, with no variability over time.17
Since 1996 in France, amoxicillin-clavulanate (AC) or cefpodoxime-proxetil (third-generation cephalosporin; 3GC) was recommended in official guidelines for treating AOM in young children.18 These choices were mainly supported by the high level of β-lactamase–producing Hi strains (about 40%) in this period.19 After the implementation of PCVs20 , 21 and the plan to reduce antibiotics use for community-acquired infections,22 the proportion of both penicillin-resistant Sp and β-lactamase–producing Hi carriage decreased, which led to change guidelines in 2011.21 For children <2 years old with AOM, the recommended treatment changed to amoxicillin given at 80–90 mg/kg/day21 according to the reduced proportion of β-lactamase–producing Hi strains and the potential for 3GC to increase the carriage and infection by extended-spectrum β-lactamase enterobacteriacae.23 However, for children with AOM and conjunctivitis, according to the role of Hi in this clinical entity, AC remained the recommended compound.21 , 24 Tympanocentesis was only recommended after a second antibiotic treatment failure.21
The dynamics of Sp and Hi antibiotic resistance justifies a sequential re-evaluation to consider changing the treatment strategies in AOM. Hence, 6 years after the change in recommended antibiotics for AOM and 4 years after PCV13 implementation, we aimed to assess this question by analyzing the trends of Sp and Hi antibiotic resistance in NP carriage in France.
MATERIALS AND METHODS
Bacteriologic data on NP carriage from 2001 to 2016 were obtained by using a cross-sectional study to monitor Sp NP carriage after PCV7 implementation.19 , 25–27 Children of both sexes, from age 6 to 24 months, with a diagnosis of AOM according to Paradise criteria and with fever and/or otalgia were enrolled by 138 pediatricians in all French metropolitan regions.28 Paradise criteria were used since the beginning of the study regardless of other diagnosis criteria for AOM to maintain the same methodology. Medical history and physical findings for each patient were obtained.
NP specimens were obtained by using cotton-tipped wire swabs as described.19 , 25–27 Swabs were placed in transport medium and transferred within 48 hours to one of the 2 centralized microbiology laboratories (Robert Debré Hospital or National Centre for Pneumococci at European Georges Pompidou Hospital in Paris, France). Morphology and standard methods were used to identify Sp and Hi strains (Hi and Mc strains were not identified between 2001 and 2005). The agar-dilution method with minimal inhibitory concentration (MIC) was used to determine susceptibility of Sp strains to penicillin. Isolates were divided into penicillin-susceptible (MIC ≤0.06 mg/L), penicillin-intermediate–resistant (MIC 0.06–2.0 mg/L) and penicillin-resistant (MIC >2 mg/L) according European Committee on Antimicrobial Susceptibility Testing breakpoints (Table version 7.1 available at http://www.eucast.org/clinical breakpoints/). Penicillin-nonsusceptible Sp (PNSP) isolates were defined as penicillin-intermediate–resistant and penicillin-resistant strains (MIC >0.06 μg/mL).
Hi and Mc strains were tested for production of β-lactamase by chromogenic cephalosporin test for ampicillin susceptibility. Hi strains were classified as ampicillin-susceptible (MIC ≤1 mg/L) and ampicillin-resistant (MIC >1 mg/L). β-Lactamase–negative, ampicillin-resistance strains were determined according to the Clinical and Laboratory Standards Institute breakpoints by a chromogenic cephalosporin test.29 Hi strains were serotyped by the slide agglutination method with specific antisera.
Data were double entered by using 4D SAS v6.4 to v12.5 (4D SAS, Le Pecq, France) and analyzed by using Stata/SE v13.1 (StataCorp, College Station, TX). We defined 5 periods according to the implementation of PCV7 and PCV13 in France20 , 30 , 31: pre-PCV7 (2001–2002), nongeneralized PCV7 (2003–2005), PCV7 (2006–2010), PCV7 to PCV13 transition (2011–2012) and PCV13 (≥2013). We used multinomial logistic regression for analysis of factors associated with penicillin-susceptible and -nonsusceptible Sp carriage as well as non-β-lactamase–producing and β-lactamase–producing Hi carriage in the PCV13 period. Clinical variables with P < 0.20 (sex, age, day care attendance modalities, siblings, fever and otalgia, otorrhea, conjunctivitis, otitis prone children, history of AOM, bilateral AOM, last antibiotic received in the last 3 months) on univariate analysis were included in the multinomial model, estimating adjusted odds ratios (aORs) and 95% confidence intervals (CIs). Quantitative data were compared by analysis of variance and qualitative data by χ2 test. All tests were 2-sided and considered significant at P < 0.05. Cuzick’s test for trend was performed over the years. Because several studies showed cross-reactive functional antibodies after PCV13 vaccination, we analyzed the vaccine serotypes as PCV13 serotype + 6C.32 , 33 Serotypes 15B and 15C were counted as only one serotype (15B/C).
The research was conducted in accordance with the Declaration of Helsinki and national and institutional standards. The study was approved by the Saint-German-en-Laye Ethics Committee. Written informed consent was obtained from parents or legal guardians.
Characteristics of Patients
Among the 12,973 children enrolled by 138 pediatricians, 9.5% (n = 1236), 15.6% (n = 2030), 32.9% (n = 4268), 13.8% (n = 1790) and 28.1% (n = 3649) were included in the pre-PCV7, nongeneralized PCV7, PCV7, transition and PCV13 periods, respectively. Table 1 presents the characteristics of patients according to the 5 periods. During the study, the proportion of children attending a DCC significantly increased from 32.5% to 54.2%. Fever and/or otalgia were less associated with AOM in the PCV13 period, whereas the proportion of AOM with conjunctivitis increased. Antibiotic prescription in the 3 months before enrollment significantly decreased (P < 0.001), with fewer oral 3GC and AC prescriptions and more amoxicillin prescriptions (P < 0.001).
Sp, Hi and Mc Carriage
Global Sp carriage slightly but significantly decreased by 18.2% after PCV13 implementation (P < 0.001; Fig. 1 and Table, Supplemental Digital Content 1, http://links.lww.com/INF/C892). The proportion of PCV7 and 6 PCV13 additional vaccine serotypes + 6C greatly decreased by 96.6% (P < 0.001) and 87.2% (P < 0.001), respectively (Fig., Supplemental Digital Content 2, http://links.lww.com/INF/C893). In the PCV13 period, nonvaccine serotypes accounted for 91.7% of Sp strains, and among Sp carriers, the main serotypes were 15B/C (14.1%), 11A (10.4%), 23B (9.3%), 15A (7.0%) and 35B (6.5%).
During the study period, Hi carriage significantly increased from 46.6 to 57.0% (P < 0.001; Fig. 2). Nontypable Hi represented 99.7% of Hi strains during the whole study.
Mc carriage slightly decreased between 2006 and 2016 from 53.2% to 49.3% (P = 0.034; Fig., Supplemental Digital Content 3, http://links.lww.com/INF/C894).
Sp Antibiotic Resistance
During the study period, the proportion of PNSP strains significantly decreased by 46.4% (P < 0.001; Fig. 1). In the pre-PCV7 period, 74.4% of these strains were PCV7 vaccine serotypes (Fig. 3). In the PCV7 period, 19A was the most frequent PNSP serotype (41.7%). In the PCV13 period, several different nonvaccine serotypes were PNSP, with 11A accounting for 16.4%, 15A for 16.3%, 35B for 16.1% and 15B/C for 8.5%.
In the PCV13 period, fully penicillin-resistant strains (MIC >2 μg/mL) accounted for 15 cases (0.8% among Sp carriers and 0.4% among all children); serotype 11A was implicated in 46.7% (n = 7) of cases, with penicillin-MIC between 3 and 8 μg/mL. The other penicillin-resistant serotypes were 9N (n = 1), 15A (n = 1), 15B (n = 1), 19F (n = 2), 29 (n = 1), 35B (n = 1) and nontypable serotype (n = 1).
Hi Antibiotic Resistance
The proportion of Hi β-lactamase–producing strains continually increased after 2012 (11.7%) to reach 23.6% in 2016 (Fig. 2). Furthermore, this proportion was significantly higher in children with than without conjunctivitis during the whole study period (18.4% versus 14.5%, P < 0.001; Fig., Supplemental Digital Content 4, http://links.lww.com/INF/C895). The proportion of β-lactamase–negative, ampicillin-resistance strains remained stable at a low level (<10%) since 2006.
Mc Antibiotic Resistance
The proportion of β-lactamase–producing strains decreased slightly during the study (P < 0.001) but remained >97%.
Risk Factors of PNSP Carriage and β-Lactamase–producing Hi Carriage in the PCV13 Period
On multinomial analysis (Table 2), DCC attendance was associated with carriage of PNSP (aOR = 2.7, 95% CI: 2.1–3.6) and β-lactamase–producing Hi (aOR = 2.4, 95% CI: 1.7–3.5). β-Lactamase–producing Hi carriage was additionally associated with conjunctivitis (aOR = 6.0, 95% CI: 4.7–7.7), siblings (aOR = 1.7, 95% CI: 1.3–2.2), history of AOM (aOR = 1.4, 95% CI: 1.1–1.9) and bilateral AOM (aOR = 1.4, 95% CI, 1.1–1.8).
Because AOM remains the leading cause of antibiotics prescriptions in children (>70%) after PCVs implementation,4 , 5 the guidelines for antibiotics treatment for this disease could have a major impact on public health. Changes in antibiotic susceptibility could lead to a modification of the guidelines. During the study period, we observed a decrease in the proportion of PNSP strains (reduced by 46.4%) and an opposite trend for β-lactamase–producing Hi strains after 2011, reaching 23.6% in 2016. Moreover, since the beginning of the study, we observed changes in AOM symptomatology, with less severe forms (decreasing rate of patients with otalgia, fever and otorrhea), which may reflect PCVs impact and the decrease of AOM caused by vaccine-type Sp.34
After PCV implementation, in France as in many countries, both pneumococcus carriage rate and the proportion of PNSP strains greatly decreased, mainly because of the decrease of PCV vaccines serotypes.1 , 27 , 35 , 36 Furthermore, in the PCV13 period, the serotypes implicated in the remaining PNSP strains completely changed, with serotypes 11A, 15A, 35B and 15BC accounting for 16.4%, 16.3%, 16.1% and 8.5%, respectively. These serotypes were not frequently isolated in the pre-PCV7 period among the PNSP strains. Moreover, in the PCV13 period, very few penicillin-resistant Sp strains were isolated (0.8%), and the main serotype was 11A. This particular serotype, contrary to serotype 19A which emerged after PCV7 implementation,19 has low AOM37 and invasive disease potential.38 Indeed, these results highlight, one more time, that the appropriate use of antibiotics is a public health priority.
By contrast, for Hi, we report an increase in carriage rate and, in the last 5 years, an increase in proportion of β-lactamase–producing strains. Because Hi carriage was associated with attending a DCC (aOR = 1.9, 95% CI: 1.7–2.2),27 the observed increase in Hi carriage we found could be attributed to the increase in frequency of children attending a DCC (increased by 40%), in accordance with the French national data.39 Concerning the trends of β-lactamase–producing Hi in the last 5 years, the explanations seem more complicated, and several factors could play a role. Among them are the increase in frequency of children attending a DCC and the change in type of antibiotics prescribed. After the antibiotic guidelines published in 2011, the prescriptions of amoxicillin sharply increased and the prescriptions of AC and 3GC greatly decreased.40 Indeed, the use of amoxicillin probably provided an ecologic advantage for β-lactamase–producing Hi. Nonetheless, the main risk factor identified for β-lactamase–producing Hi strains among all children was conjunctivitis (aOR = 6.0, 95% CI: 4.7–7.7).41 During the last 3 years for AOM without conjunctivitis, the percentage of β-lactamase–producing strains remains below 20%.
Mc is the third bacterial species implicated in AOM when using middle-ear fluid culture.2 , 3 In France, although Mc strains are frequently isolated from NP flora, the proportion of AOM because of Mc is very low (<8%).42 However, polymerase chain reaction techniques have increased the proportion of cases in which Mc is found, particularly for children with otorrhea and/or mixed infections or recurrent cases.17 , 43 Although this otopathogen is almost always resistant to amoxicillin through β-lactamase production,17 AOM caused by Mc is described as less severe, extremely rarely complicated (mastoiditis, bacteremia or meningitis) and more likely to resolve spontaneously without antibiotic treatment.2 , 44 For these reasons, many national guidelines (US, UK, Canada, Israel and France) do not consider that M. catarrhalis should be a target for first-line antibiotic treatment in AOM in the same way as Sp and Hi.8 , 9 , 21 , 45 , 46
The main limitation in our study is the use of microbiologic culture of samples from the nasopharynx instead of from middle-ear fluid to analyze resistance of bacterial strains involved in AOM. NP cultures are imperfect to predict individually the etiology of AOM.12 , 13 As shown previously by Kaur et al,14 for epidemiologic studies, NP cultures, when samples are collected at onset of AOM, may predict antibiotic susceptibility of middle-ear fluid. Furthermore, relative change in the prevalence of vaccine-serotype Sp carriage has been described as a predictor of relative change in incidence of AOM because of vaccine-serotypes Sp.15 Indeed, pneumococcus implicated in AOM could differ from those isolated from NP flora. Shouval et al37 addressed the relationship between carriage of individual serotypes and AOM (positive middle-ear fluid culture) and for the serotypes 11A, 15A, 35B and 15B/C did not find any significant positive association. If in some regions, tympanocentesis with culture continue to be performed,47 bacteriologic samples of middle-ear fluid are not routinely obtained in France.48 Indeed, tympanocentesis is mainly used for complicated AOM (unresponsive cases, recurrent cases, otorrhea or with underlying conditions) with probably an overrepresentation of resistant strains.
Sp and Hi NP carriage is a dynamic process mainly subject to antibiotic use, vaccination and interactions between bacterial species in the nasopharyngeal microbiota.16 , 49 , 50 The continuous modification of antibiotic resistance among these 2 otopathogens justifies ongoing NP carriage surveillance and regular update. With this study and the changes observed, the question is the relevance of the treatment strategies in AOM. Although we observed an increase in β-lactamase–producing Hi carriage, amoxicillin should remain the drug of choice for first-line AOM because of its better tolerability and the lesser ecological impact of this compound as compared with AC or 3CG.23
For children with AOM and conjunctivitis, because of the role of Hi and the frequency of β-lactamase–producing Hi carriage, the treatment should remain AC, especially for children attending a DCC.
We thank all pediatricians who participated in the study: C. Abt-Nord, M.-J. Aim-Mille, D. Allain, M. Amzallag, P. André, I. Aubier, P. Bakhache, J. Baron, B. Baszanger, C. Batard, G. Beley, M. Benani, C. Bensoussan-Ambacher, E. Billard, L. Billet, J.-P. Blanc, M.-J. Bodin, E. Boez, B. Bohe, J. Bouglé, F. Bouillot, B. Broussin, J.-L. Cabos, P. Camier, F. Ceccato, A. Chevé, D. Clavel, C. Claverie, R. Cohen, L. Coicadan, F. Corrard, L. Cret, G. d’Acremon, N. D’Ovidio-Panis, B. de Brito, F. De Grenier, P. Deberdt, I. Defives, A. Delatour, J.-F. Delobbe, V. Derkx, V. Desvignes, M. Dogneton, I. Donikian-Pujol, C. Douer-Fernando, M. Dubosc, J. Ducellier, C. Dumont, A. Elbez, J. Elbhar, N. Elkhoury, C. Ferte-Devin, J.-M. Fiorini, D. Garel, J.-L. Gasnier, A. Gasser, B. Gaudin, N. Gelbert-Baudino, P. Gembara, C. Georgeot, M. Gerardin, M. Giorno, M. Goldrey, R. Gorge, J. Gosselin, C. Guiheneuf, J.-L. Guillon, J.-F. Hassan, A. Hayat, H. Hollenberg, P. Huguet, P. Hugot, M. Hunin, A. Kalindjian, K. Kassmann, Z. Klink, M. Koskas, C. Lastman-Lahmi, H. Le Scornet, M.-C. Lemarchand, J.-C. Lévêque, J. Levy, M. Levy, D. Livon, N. Maamri-Belaroussi, C. Magendie, I. Martins, M.-O. Mercier-Oger, C. Messica, F. Meunier, A.-S. Michot, J. Miclot, P. Migault, I. Nave, M. Navel, J.-F. Nicolas, M.-O. Oger, J. Ohayon, J.-C. Oilleau, A. Pappo, F. Paratte, J. Peguet, C. Perrin, C. Petit, B. Pinçant, O. Pinard, A. Piollet, J.-F. Pujol, M.-T. Pujol, N. Ramos, S. Ravilly, Y. Regnard, M. Robert, O. Romain, S. Romano, M.-C. Rondeau, B. Saade, C. Salinier-Rolland, C. Schlemmer, E. Seror, G. Sivelle, D. Somerville, N. Temam-Basse, J.-M. Thiron, F. Thollot, J. Toledanno, R. Touitou, C. Turberg-Romain, J. Vaugeois, F. Vie Le Sage, J.-L. Vuillemin, A. Werner, R. Wisnewsky, A. Wollner, C. Wollner, C. Ythier. We thank M. Boucherat, M. Fernandes, I. Ramay, D. Menguy, C. Prieur, A. Prieur and E. Sobral and N. Hamouda for technical assistance.
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