Pneumococcal Acute Otitis Media Aom

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1

2 Pneumococcal acute otitis media in infants and children in central

3 Romania, 2009–2011: microbiological characteristics and potential

4 coverage by pneumococcal conjugate vaccines

5 O. Falup-Pecurariu Q1

a, E. Leibovitz b,*, A. Mercas a, L. Bleotu a, C. Zavarache a, N. Porat b,

6 R. Dagan b, D. Greenberg b

7 a Department of Pediatrics, Children’s Hospital, Faculty of Medicine, Transilvania University, Brasov, Romania

8 b Pediatric Infectious Disease Unit, Soroka University Medical Center, Ben-Gurion University, Beer-Sheva, Israel Q2

9

10

11 1. Introduction

12 Acute otitis media (AOM) is the most frequent bacterial disease

13 of childhood, affecting millions of children worldwide and

14 remaining a major public health problem.1–3 The most common

15 causative agents in AOM are Streptococcus pneumoniae, non-

16 typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, and

17 Streptococcus pyogenes.4–8 Together, S. pneumoniae and NTHi

18 account for 60–80% of the AOM pathogens.9 Antibiotic resistance

19 is high in pneumococcal AOM, with penicillin- and amoxicillin-

20 non-susceptible strains accounting for 30–70% of cases.10–16 The

21 most commonly encountered S. pneumoniae serotypes in AOM are

22 6A, 6B, 14, 19A, 19F, and 23F.15,16 The highest antibiotic non-

23 susceptibility is found in vaccine serotypes 6A, 6B, 9 V, 14, 19A,

24 19F, and 23F.11,12,15,16

25 Information on antibiotic resistance patterns and serotype

26 distribution of S. pneumoniae isolates in infants and young children

27 in Romania is limited. In a multinational study, 42% of

28 S. pneumoniae AOM isolates from Romania were intermediately

29 or fully resistant to penicillin.17 In three studies investigating

30 pneumococcal mucosal and invasive disease isolates in

International Journal of Infectious Diseases xxx (2013) xxx.e1–xxx.5

A R T I C L E I N F O

Article history:

Received 23 November 2012

Received in revised form 26 December 2012

Accepted 2 February 2013

Corresponding Editor: Eskild Petersen,

Aarhus, Denmark

Keywords:

Acute otitis media

Streptococcus pneumoniae

Antibiotic resistance

Children

S U M M A R Y

Objective: To assess the epidemiological and microbiological characteristics of pneumococcal acute otitis

media (AOM) in children in Brasov, Central Romania, before the introduction of pneumococcal conjugate

vaccine (PCV) into the routine national immunization program.

Methods: All AOM patients aged <5 years who underwent tympanocentesis or presented with purulent

otorrhea of _24 h duration during 2009–2011 were enrolled.

Results: Two hundred and twelve consecutive AOM patients had a middle ear fluid (MEF) culture

performed; 99 (46.6%) episodes occurred in patients <12 months of age. One hundred and eleven (52.4%)

episodes were culture-positive. Tympanocentesis was performed in 142 patients and spontaneous

otorrhea cultures in 70 patients. Overall, 114 pathogens were recovered: Streptococcus pneumoniae was

the most common isolate (81 isolates, 70.3% of all culture-positive episodes), followed by non-typeable

Haemophilus influenzae (26, 20.7%), Streptococcus pyogenes (5, 4.5%), and Moraxella catarrhalis (2, 1.8%).

Antibiotic susceptibility and serotyping were performed for 48 (59.3%) S. pneumoniae isolates: 45 (93.8%)

were non-susceptible to penicillin (minimal inhibitory concentration (MIC) _2.0 mg/ml in 24, 53.3%) and

37 (77.1%) isolates had ceftriaxone MIC values _0.5 mg/ml (16 with MIC >2.0 mg/ml). S. pneumoniae

non-susceptibility rates to trimethoprim–sulfamethoxazole, erythromycin, and clindamycin were

75.0%, 58.3%, and 35.4%, respectively. All isolates were susceptible to chloramphenicol. Multidrug

resistance was found in 33 (68.7%) isolates. The most common S. pneumoniae serotypes were 19F (14,

29.2%), 6B (8, 16.7%), 23F (8, 16.7%), and 14 (6, 12.5%). Serotype 19A was found in three (6.2%) patients

and 6A in two (4.1%). Non-PCV13 serotypes represented six (12.6%) of all serotypes (four of them nonsusceptible

to penicillin). Thirty-six (75.0%) isolates were potentially covered by PCV7, 37 (77.0%) by

PCV10, and 42 (87.5%) by PCV13.

Conclusions: (1) S. pneumoniae was the most prevalent pathogen, with frequent antibiotic resistance and

multi-resistance patterns; (2) most pneumococcal AOM and multidrug-resistant episodes could be

prevented by PCVs.

_ 2013 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

* Corresponding author.

G Model

IJID 1656 1–5

Please cite this article in press as: Falup-Pecurariu O, et al. Pneumococcal acute otitis media in infants and children in central Romania,

2009–2011: microbiological characteristics and potential coverage by pneumococcal conjugate vaccines. Int J Infect Dis (2013), http://

dx.doi.org/10.1016/j.ijid.2013.02.002

Contents lists available at SciVerse ScienceDirect

International Journal of Infectious Diseases

jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/ijid

1201-9712/$36.00 – see front matter _ 2013 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

http://dx.doi.org/10.1016/j.ijid.2013.02.002

31 HIV-negative and HIV-positive infants and children in northeast-

32 ern Romania, high rates of recovery of serotypes 19A and 23F and

33 of multidrug-resistant (MDR) organisms were reported.18–20

34 Recently, high rates of colonization with S. pneumoniae (reaching

35 71% in children 13–24 months of age) accompanied by high

36 resistance rates to most of the commonly used antibiotic drugs

37 were reported among healthy and sick infants and children

38 <5 years old in central Romania.21

39 Active and continuous surveillance of the microbiology and

40 antibiotic susceptibility patterns of AOM pathogens in Romania is

41 important, particularly during the period preceding the introduc-

42 tion of pneumococcal conjugate vaccine (PCV). The aims of the

43 present study were to assess: (1) the overall distribution of

44 otopathogens and their antibiotic resistance patterns; and (2) PCV

45 coverage of S. pneumoniae middle ear fluid (MEF) isolates in Brasov,

46 central Romania, during 2009–2011.

47 2. Patients and methods

48 We conducted a prospective epidemiological study between

49 January 1, 2009 and December 31, 2011, at the Children’s Hospital

50 of Brasov, in the central part of Romania. Brasov has a population of

51 400 000 inhabitants and the hospital is the only medical center

52 providing medical care for infants and children in the city. The

53 study protocol was approved by the institutional review board of

54 the University of Transilvania, Brasov. Informed consent was

55 obtained from the legal guardians of all children.

56 2.1. Patients and procedures

57 The study patients were infants and children aged <5 years

58 diagnosed with AOM by a pediatrician, family physician, or

59 otolaryngologist. The diagnosis was done when the patients

60 presented with: (1) symptoms and physical findings consistent

61 with AOM (fever, irritability, tugging of the ear, and redness and

62 bulging of the tympanic membrane with blurring of its anatomic

63 landmarks); (2) acute illness lasting _7 days. Bulging of the

64 tympanic membrane (in addition to clinical symptoms of AOM)

65 was present in all cases where a tympanocentesis was performed.

66 Culture specimens were obtained by either tympanocentesis or

67 collection of pus from draining the ears (if lasting _24 h before

68 enrollment). Patients with tympanostomy tubes were excluded

69 from the study. In all episodes, we collected information on the

70 patient’s age, sex, and ethnicity, the type of specimen (tympano-

71 centesis or draining pus), and the patient’s AOM history and recent

72 antibiotic treatment. Data were obtained from the medical records

73 of the hospital, clinical medical chart, or parent interview. None of

74 the patients had been immunized with a PCV before enrollment.

75 Tympanocentesis was performed by the study otolaryngologist

76 (AM) as previously described.7,12

77 2.2. Bacteriology

78 Swabs of MEF aspirates were placed in MW173 Amies transport

79 medium (Transwab; Medical Wire and Equipment), plated

80 immediately on trypticase agar containing 5% sheep blood and

81 5 mg/ml gentamicin, and on chocolate agar, and incubated at 35 8C

82 for 48 h in a 5% enriched CO2 atmosphere. Identification,

83 serotyping, and testing of antimicrobial susceptibility to penicillin

84 and ceftriaxone (by E-test, PDM Epsilometer, AB Biodisk, Solna,

85 Sweden) and erythromycin, clindamycin, chloramphenicol, and

86 trimethoprim–sulfamethoxazole (TMP–SMX) (by disk diffusion)

87 were performed as described elsewhere, in accordance with the

88 Clinical and Laboratory Standards Institute (CLSI) recommenda-

89 tions.22 Non-susceptibility to _3 antibiotic classes was considered

90 multidrug resistance (MDR). Serotyping was done by quellung

91 reaction.23 The organisms were sub-cultured and stored at _70 8C

92 at the Microbiology Laboratory of the Children’ Hospital of Brasov

93 and were further transported by air to the Pediatric Infectious

94 Disease Unit Laboratory of the Soroka University Medical Center,

95 Beer-Sheva, Israel, where all antimicrobial susceptibility testing

96 and serotyping was carried out.

97 S. pneumoniae isolates were considered susceptible to penicillin

98 if the minimal inhibitory concentration (MIC) values were

99 _0.06 mg/ml, intermediate if penicillin MIC values were between

100 0.125 mg/ml and 1.0 mg/ml, and resistant if MIC values were

101 _2.0 mg/ml. Ceftriaxone intermediate resistance was defined by

102 MICs values between 0.5 and 1.0 mg/ml, and high resistance by

103 MICs values >2.0 mg/ml.

104 2.3. Statistical analysis

105 Data were recorded using Microsoft Access office software. The

106 statistical analysis was performed using SPSS 17.0 software.

107 Contingency table analysis for comparing rates between un-

108 matched samples was performed using the Chi-square test or

109 Fisher’s exact test, as appropriate. The Student independent

110 samples t-test was used to compare continuous variables. The

111 percentages of serotype coverage were calculated and compared

112 between PCV7 (serotypes 4, 6B, 9 V, 14, 18C, 19F, and 23F), PCV10

113 (PCV7 plus serotypes 1, 5, and 7F), and PCV13 (PCV10 plus

114 additional serotypes 3, 6A, and 19A). All tests were considered

115 significant if p-values were <0.05.

116 3. Results

117 During the study period, 212 consecutive infants and young

118 children <5 years of age were enrolled. There were 120 (56.6%)

119 males. The mean age (_ standard deviation) was 18.0 _ 14.2 months.

120 Ninety-nine (46.6%) episodes occurred in patients <12 months old

121 and 136 (64.2%) episodes occurred in children <2 years old. One

122 hundred and eleven samples (52.4%) were culture-positive. Children

123 with culture-positive MEF were older than children with culture-

124 negative MEF (20.6 _ 15.2 months vs. 15.4 _ 12.6 months, p = 0.008).

125 Tympanocentesis was recorded in 142 patients and spontane-

126 ous otorrhea in 70 patients. No differences were recorded in the

127 proportions of spontaneous perforation between children with

128 culture-positive and children with culture-negative MEF (38/111,

129 34.2% vs. 32/101, 31.7%, p = 0.7).

130 A total of 114 isolates (76 from tympanocentesis and 38 from

131 spontaneous otorrhea specimens) were recovered. S. pneumoniae

132 was the most common isolate (81 isolates, 78 episodes, 71.1% of all

133 pathogens recovered and 70.3% of all culture-positive episodes),

134 followed by NTHi (26, 23; 22.8% and 20.7%), Streptococcus

135 pyogenes (5, 5; 4.4% and 4.5%) and Moraxella catarrhalis (2, 2;

136 1.8% and 1.8%) (Table 1). In three patients (1.4% of all episodes),

137 both S. pneumoniae and NTHi were isolated. No differences were

138 recorded in the percentages of S. pneumoniae recovered from

Table 1

Acute otitis media microbiology of 212 episodes (111 culture-positive) during

2009–2011

Pathogen No. of episodesa

Streptococcus pneumoniae 78 (70.3)

Haemophilus influenzae 23 (20.7)

Streptococcus pyogenes 5 (4.5)

Moraxella catarrhalis 2 (1.8)

S. pneumoniae + H. influenzae 3 (2.7)

Culture-positive 111

Culture-negative 101

Total 212

a The percentage of all culture-positive episodes is given in parenthesis.

O. Falup-Pecurariu et al. / International Journal of Infectious Diseases xxx (2013) xxx.e1–xxx.e5 e2

G Model

IJID 1656 1–5

Please cite this article in press as: Falup-Pecurariu O, et al. Pneumococcal acute otitis media in infants and children in central Romania,

2009–2011: microbiological characteristics and potential coverage by pneumococcal conjugate vaccines. Int J Infect Dis (2013), http://

dx.doi.org/10.1016/j.ijid.2013.02.002

139 tympanocentesis compared with those isolated from spontane-

140 ous otorrhea (55/76, 72.4% vs. 26/38, 68.4%, p = 0.7). No

141 differences were recorded in the mean age at diagnosis of

142 patients with S. pneumoniae AOM compared with patients with

143 AOM caused by other etiologic agents (18.16 _ 7.08 vs.

144 24.59 _ 18.37 months, p = 0.1).

145 Reliable information on prior antibiotic treatment (during the

146 24 h preceding AOM diagnosis) was available for 68/111 (60.2%)

147 culture-positive patients; 29/68 (42.6%) culture-positive patients

148 received previous antibiotic treatment compared with 34/101

149 (33.7%) culture-negative patients (p = 0.24). The representation of

150 S. pneumoniae was higher in patients previously untreated with

151 antibiotics compared with patients treated with antibiotics (28/39,

152 71.7% vs. 14/29, 48.3%, p = 0.03).

153 Antibiotic susceptibility testing and serotyping were performed

154 for 48/81 (59.3%) S. pneumoniae isolates. Forty-five (93.8%)

155 S. pneumoniae isolates were non-susceptible to penicillin; 24

156 (53.3%) had MIC values _2.0 mg/ml. Six isolates had a penicillin

157 MIC value of 16.0 mg/ml, one had a MIC of 8.0 mg/ml, and three had

158 a MIC of 4.0 mg/ml. Thirty-seven (77.1%) isolates had ceftriaxone

159 MIC values _0.5 mg/ml (32.0 mg/ml for five isolates, 4.0 mg/ml for

160 nine isolates, 2.0 mg/ml for one isolate, and 1.0 mg/ml for one

161 isolate). The non-susceptibility rates to TMP–SMX, erythromycin,

162 and clindamycin were 36/48 (75%), 28/48 (58.3%), and 17/48

163 (35.4%), respectively. All isolates were susceptible to chloram-

164 phenicol. Resistance to _1 antibiotic class was found in 44 (91.7%)

165 and MDR in 33 (68.7%) S. pneumoniae isolates. No differences were

166 recorded in the resistance patterns to antibiotics of S. pneumoniae

167 recovered from tympanocentesis versus the isolates recovered

168 from culture of spontaneous otorrhea.

169 The most common S. pneumoniae serotypes were: 19F (14,

170 29.2%), 6B (8, 16.7%), 23F (8, 16.7%), and 14 (6, 12.5%) (Table 2).

171 Serotype 19A was found in three (6.2%) patients and 6A in two (4.1%)

172 patients. The non-PCV13 serotypes represented six (12.6%) of all

173 serotypes, and four of them were non-susceptible to penicillin. Six

174 (75%) of the eight serotype 23F isolates had a penicillin MIC of

175 16.0 mg/ml and a ceftriaxone MIC value of 32.0 mg/ml; 3/14 (21.4%)

176 serotype 19F isolates had a penicillin MIC value of 4.0 mg/ml and one

177 had a penicillin MIC value of 8.0 mg/ml. The MDR isolates included,

178 in descending order, serotypes 19F, 6B, 23F, 19A, and 6A. Of the 14

179 serotype 19F isolates, 13 (92.9%) were non-susceptible to penicillin,

180 erythromycin, and TMP–SMX, and 9/13 (69.1%) also to clindamycin.

181 All eight serotype 6B isolates were non-susceptible to penicillin,

182 erythromycin, TMP–SMX, and clindamycin. All eight serotype 23F

183 isolates were non-susceptible to penicillin, erythromycin, and TMP–

184 SMX, and 5/8 (62.5%) also to clindamycin. All three serotype 19A

185 isolates were non-susceptible to penicillin, erythromycin, and TMP–

186 SMX, and 2/3 (66.7%) also to clindamycin. One of the two serotype 6A

187 isolates was non-susceptible to penicillin, erythromycin, TMP–SMX,

188 and clindamycin.

189 Of the 48 tested S. pneumoniae serotype isolates, 36 (75.0%)

190 are included in PCV7, 37 (77.0%) in PCV10, and 42 (87.5%) in

191 PCV13.

192 4. Discussion

193 The present study aimed to provide baseline data on the

194 contribution of S. pneumoniae to the etiology of AOM in Romania.

195 We also analyzed the resistance patterns to antibiotics and the

196 serotype distribution of this pathogen before the introduction of

197 universal PCV immunization in Romania.

198 Our main findings were: (1) infants and young children with

199 culture-positive AOM were older compared with those with

200 culture-negative AOM; (2) S. pneumoniae was the dominant

201 etiologic agent, being isolated in 74.3% of the culture-positive

202 patients; (3) most of the isolates were resistant to penicillin,

203 erythromycin, and TMP–SMX, and MDR was very common;

204 (4) the current PCVs could potentially prevent most of the

205 pneumococcal AOM in the region. The present study may

206 provide a basis for follow-up and monitoring of AOM etiology

207 and resistance patterns after the introduction of PCV into the

208 national program.

209 In the Brasov area, we recently reported that of 205

210 pneumococcal nasopharyngeal isolates obtained from infants

211 and children <5 years of age (attending daycare centers,

212 immunization clinics, or visiting the pediatric emergency room

213 of the hospital for different acute illnesses, as well as healthy

214 patients admitted to the surgery department for elective

215 procedures), 83% were non-susceptible to penicillin and 18%

216 were non-susceptible to ceftriaxone, of which 40.5% and 16% were

217 highly-resistant to penicillin and ceftriaxone, respectively.21 The

218 non-susceptibility rates to erythromycin, TMP–SMX, tetracycline,

219 and clindamycin were also high (>50% for each antibiotic) and

220 MDR was found in 67% of isolates. The most common pneumo-

221 coccal serotypes isolated were 23F, 6B, 19F, 14, 6A, and 19A, and

222 the potential coverage by PCV7, PCV10, and PCV13 was 66%, 74%,

223 and 80%, respectively. This is consistent with the AOM findings in

224 the current study.

225 The major limitation of this study derives from the small

226 number of pneumococcal isolates evaluable for antibiotic

227 susceptibility testing and serotyping. On the other hand, the data

228 presented here from MEF cultures performed in AOM patients

229 diagnosed and treated at the Brasov Children’s Hospital are

230 additional to previously published information on the pneumo-

231 coccal carriage in patients enrolled from daycare centers and

232 immunization clinics in the city of Brasov and also from the

233 emergency room and surgery department of the hospital (which is

234 the only referral pediatric hospital in the whole area and is the

235 only site where MEF and nasopharyngeal cultures are per-

236 formed).21 Therefore, we are convinced that these two studies

237 from Brasov provide an up-to-date and reliable picture of the local

238 pneumococcal burden in infants and young children and the

239 antibiotic susceptibility, serotype distribution, and potential

240 serotype coverage by PCVs in the city of Brasov and the

241 surrounding areas. At the present time, additional limited data

242 from nasopharyngeal and MEF pneumococcal isolates obtained

243 from infants and young children are available from the

244 northeastern (Iasi) and southern (Bucharest) areas of the country

245 and provide a similar picture in terms of colonization burden,

246 extremely high antibiotic resistance rates, serotype distribution,

247 and potential PCV coverage.17–21,24

248 The presented data, together with the additional data from

249 northeastern Romania, raise major concerns regarding the

250 unskilled used of antibiotics in this country, leading to high

Table 2

Streptococcus pneumoniae serotype distribution (in decreasing frequency) for 48

acute otitis media episodes

Serotype No. episodes (%) MDR

19F 14 (29.2) 13 (92.9%)

23F 8 (16.7) 8 (100%)

6B 8 (16.7) 8 (100%)

14 6 (12.5) -

19A 3 (6.2) 3 (100%)

6A 2 (4.1) 1 (50%)

22F 2 (4.1) -

9V 1 (2.1) -

34 1 (2.1) -

9A 1 (2.1) -

7F 1 (2.1) -

Omni-negative 1 (2.1) -

Total 48 33 (68.7%)

MDR, multiple drug resistance.

O. Falup-Pecurariu et al. / International Journal of Infectious Diseases xxx (2013) xxx.e1–xxx.e5 e3

G Model

IJID 1656 1–5

Please cite this article in press as: Falup-Pecurariu O, et al. Pneumococcal acute otitis media in infants and children in central Romania,

2009–2011: microbiological characteristics and potential coverage by pneumococcal conjugate vaccines. Int J Infect Dis (2013), http://

dx.doi.org/10.1016/j.ijid.2013.02.002

resistance. Furthermore, the high rate of resistance is of concern in

252 terms of the efficacy of current antimicrobial agents in the

253 treatment of AOM.

254 Faced with the alarming resistance data presented in this

255 study, an intervention program including a major reduction in

256 antibiotic use, combined with introduction of routine vaccination

257 with PCVs, is much needed in Romania. In France, Cohen et al.25

258 clearly showed that the implementation of a national program of

259 reduction of inappropriate antibiotic use markedly contributed to

260 the efficacy of PCV7 in reducing the carriage of penicillin-non-

261 susceptible pneumococci in children with AOM. In a study

262 evaluating the association between antibiotic use in the commu-

263 nity and the increase in antibiotic-resistant S. pneumoniae

264 carriage in Bedouin children aged <5 years in southern Israel

265 from 1998 to 2005, Greenberg et al.26 examined all the antibiotic

266 prescriptions provided from two community primary pediatric

267 clinics and reported a decrease by 19% in the total annual

268 prescription rates, mainly as a result of a reduction in amoxicillin–

269 clavulanate prescriptions. Oral cephalosporins, erythromycin,

270 and penicillin prescription rates decreased significantly as well,

271 but azithromycin prescription rates increased significantly during

272 the study period. In parallel, the authors analyzed the

273 S. pneumoniae nasopharyngeal carriage in healthy children

274 <5 years old from the same communities and were able to

275 demonstrate an increase in the proportion of nasopharyngeal

276 S. pneumoniae with penicillin MICs _1.0 mg/ml from 8% to 21% and

277 significant increases in resistance to clindamycin, erythromycin,

278 and tetracycline and also in multidrug resistance. The authors

279 suggested an association between the increased carriage of MDR

280 S. pneumoniae and the increased azithromycin consumption, and

281 cautioned that a reduction in the total antibiotic use may not be

282 sufficient as long as antibiotics with a high potential for the

283 promotion of multidrug resistance, like azithromycin, continue to

284 be used widely.26

285 The introduction of the 7-valent conjugate PCV (PCV7)

286 had a major role in the reduction of invasive and mucosal

287 disease rates caused by S. pneumoniae and of the antimicrobial

288 resistance of the isolated organisms, and also, although less

289 impressive, in the reduction of nasopharyngeal colonization

290 and AOM cases caused by this pathogen.27–32 In our study, the

291 good coverage of overall pneumococcal serotypes and also of

292 MDR pneumococcal isolates by all PCVs, and in particular by

293 PCV13 (87.5% and 100%, respectively), are important and

294 encouraging findings.

295 PCV7 was registered in Romania in September 2007, but is not

296 yet included in the routine immunization program for Romanian

297 infants and children. Initiation of a national immunization

298 program is urgently needed in order to achieve a reduction in

299 pneumococcal disease in general and of antibiotic-resistant and

300 MDR pneumococcal AOM in particular.

301 Acknowledgements

302 This study was supported by a European Society of Infectious

303 Diseases (ESPID) Research Grant (2010).

304 Conflict of interest: Prof. Ron Dagan has received grants/research

305 support from Berna/Crucell, Pfizer, MSD, and Protea; has been a

306 scientific consultant for GlaxoSmithKline, Pfizer, NASVAX, and

307 MSD and a speaker for Berna/Crucell, GlaxoSmithKline, and Pfizer;

308 he is a shareholder in Protea/NASVAX. All other authors report no

309 conflict of interest.