Until the end of the past century, most studies of acute otitis media (AOM) identified Streptococcus pneumoniae as the most common etiologic agent of this disease.1 Serotypes included in the heptavalent pneumococcal conjugate vaccine (PCV7) were detected in the middle ear fluid (MEF) of most children with AOM, suggesting a potential role of this vaccine in reducing AOM incidence.2 However, after several years of PCV7 use, replacement of PCV7 serotypes by nonvaccine serotypes occurred and the incidence of pneumococcal AOM nearly returned to previous values.3 In particular, serotypes 19A and 3 were found to be increasing. Because these serotypes were included in the 13-valent pneumococcal vaccine (PCV13), it was expected that administration of PCV13 would be effective in reducing pneumococcal AOM incidence.4 However, also after the introduction of PCV13, direct and indirect evidence of serotype replacement in invasive pneumococcal disease (IPD) was observed.5,6 Although monitoring the dynamics of S. pneumoniae serotypes leading to AOM is extremely important in evaluating PCV13 efficacy, the data regarding pneumococcal serotypes in AOM after PCV13 implementation are scarce. The main aim of this study was to determine which pneumococcal serotypes could be detected in the MEF of children with AOM complicated by spontaneous tympanic membrane perforation (STMP) living in the greater Milan area, Italy, where since 2011 ≥90% of the children have received the PCV13 vaccine in the first year of life.
MATERIALS AND METHODS
In this study, MEF specimens from children with AOM associated with STMP consecutively seen by 6 primary care pediatricians in the greater Milan area between April 1, 2015, and March 31, 2016 were evaluated. Children with tympanostomy tubes, craniofacial abnormalities or chronic middle ear conditions (including chronic tympanic membrane perforation), who had primary or secondary immunodeficiency, who had dysmorphic or genetic syndromes, who had received antibiotics in the previous 2 weeks and who were receiving topical (ie, in the ear and/or the nose) antibiotic treatments were excluded. The diagnosis of AOM with STMP was confirmed by an experienced pediatrician and validated otoscopist and was based on acute symptoms (ie, fever, irritability or earache) lasting ≤3 days and otorrhea within 12 hours of the STMP. MEF was obtained after the bulk of the otorrhea fluid had been removed and the ear canal had been cleaned with a dry cotton swab. Under direct otoscopic visualization, the remaining MEF was collected from very near the perforation using an extra-thin flexible wire swab (eNAT transport and preservation medium, Copan, Brescia, Italy). If both tympanic membranes had perforated, only 1 was considered, and 1 swab was taken from each patient.
The patients’ clinical histories were reviewed, and their data were entered into a computerized database. The children who, at the time of enrollment, had a history of ≥3 episodes of AOM in the previous 6 months or ≥4 in the previous year were considered as having recurrent AOM. The patients’ pneumococcal vaccination status was established by consulting the official vaccination chart issued by the Vaccination Service of the Lombardia Region. The pneumococcal immunization schedule recommended by the Italian Ministry of Health includes 3 doses of PCV in the first year of life; PCV7 was used from September 2002, to November 2010, and was later replaced by PCV13. The patients were considered fully vaccinated if the recommendation had been fulfilled by the time of enrollment, and patients were considered not fully vaccinated if they had started but not completed the vaccine schedule. Because the latter group comprised only 1% of the enrolled subjects, it was not compared with the groups of fully vaccinated or unvaccinated children.
The study was approved by the Ethics Committee of Milan Area B, Italy, and written informed consent was obtained from the patients’ parents or legal guardians.
Bacterial genomic DNA was extracted from the swabs using a NucliSENS easyMAG automated extraction system (BioMeriéux, Bagno a Ripoli, Florence, Italy) and tested for the autolysin-A-encoding (lytA) and wzg (cpsA) genes of S. pneumoniae using real-time polymerase chain reaction (PCR) as previously described.7 The level of detection of the test was 16 genome copies, and each sample was tested in triplicate and considered positive if at least 2 of the 3 tests revealed the presence of both genes. The real-time PCR-negative specimens were also tested for the presence of an RNase P-encoding gene to exclude PCR inhibition and DNA extraction failure. All of the positive cases were serotyped using primers and probes designed on the basis of the GenBank database sequences (http://www.ncbi.nlm.nih.gov) of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C/D, 7 F, 8, 9 V, 10A/B, 11A/D, 12A/B/F, 14, 16F, 17F, 18C, 19A, 19 F, 20, 21, 22A/F, 23A, 23B, 23F, 24A/B/F, 29, 33A/F, 35B, 35F and 38 and were synthesized by TIB Molbiol (Genoa, Italy) as previously described.7
The clinical characteristics of all patients and the prevalence of pneumococcal serotypes were summarized, for different age groups and vaccination status, using absolute numbers and percentages. All the statistical analyses were performed using R software, version 3.2.2.
A total of 177 children (103, 58.2%, males; mean age ± standard deviation, 3.5 ± 2.7 years) were enrolled. Among them, 162 (91.5%) children were fully vaccinated with PCV7 or PCV13. A history of recurrent AOM was present in 100 (56.5%) of the children. Table 1 summarizes the prevalence of pneumococcal serotypes in the MEF of children with AOM associated with STMP according to age group and pneumococcal vaccination status. S. pneumoniae was identified in the MEF of 48 (27.1%) subjects. The prevalence was higher in younger patients than in older patients: 24 (36.9%) of those <2 years old, 14 (20.0%) of those 2–4 years old and 10 (23.8%) of those ≥5 years old. Among the S. pneumoniae-positive cases, 23/24 (95.8%) and 13/14 (92.9%) in the groups of children <2 years and 2–4 years old, respectively, were fully vaccinated with PCV13. Of children ≥5 years old, 6/10 (60%) had received PCV7 and 3/10 (30%) had received PCV13. Serotyping of S. pneumoniae revealed that in most of the cases (37/48, 77.1%) serotypes not included in PCV13 were present, independent of age and the previous history of recurrent AOM: 13 (68.4%) in children with single AOM and 24 (82.8%) in children with recurrent AOM (P = 0.30). Serotypes 15A/F, 11 A/D and 24A/B/F were the most common. Serotypes included in PCV13—mainly 1, 3 and 19F—were identified in 8 (33.3%) children <2 years old, 4 (28.6%) children 2–4 years old and 3 (30%) children ≥5 years old. All but 1 child (<2 years old) had received the PCV available when they were in the first year of life.
Considering the difficulties of obtaining MEF by means of tympanocentesis, spontaneous otorrhea provides an easier method to approach the etiology of AOM. This study confirms that S. pneumoniae is one of the pathogens more commonly associated with the development of this disease even when it is complicated by STMP.8,9 Moreover, it shows that, despite full immunization with PCV13, children can develop AOM due to serotypes included in the vaccine they received. This is not surprising because it has been shown that the protection offered by PCVs against AOM is lower than that observed for IPD, particularly when serotypes such as serotype 3—for which very high IgG antibody concentrations are needed to ensure protection—are the infecting agents.10 However, the most important finding of this study is the evidence that, approximately 5 years after the introduction of PCV13 in greater Milan in Italy, most of the pneumococcal cases of AOM with STMP are associated with serotypes not included in the vaccine. Although there are no data indicating which serotypes were associated with AOM complicated by STMP in the prevaccination period in this area, studies on IPD11 and carriage12 in Italy showed that vaccine-related serotypes were those most frequently detected in prevaccination era. This finding suggests that a significant replacement phenomenon has developed, reducing the potential effect of PCV13 on AOM incidence.
The role of non-PCV13 serotypes as causes of pneumococcal disease has already been reported in studies of IPD5,6 and AOM.4,13 However, the prevalence of AOM with STMP due to these serotypes that was identified in this study is the highest ever reported. In this respect, our data are different from those reported by other authors that showed only a limited role of non-PCV13 serotypes in the etiology of AOM13 as well as those reported by Ben-Shimol et al4, who found only a marginal, not statistically significant, replacement phenomenon. These differences might be explained by the fact that this study, in contrast to the others, was carried out several years after the introduction of PCV13 and enrolled children living in an area with very high PCV13 vaccination coverage. On the other hand, the 6 primary care pediatricians who participated in the study worked in the largest Italian region, far 1 from the other and microepidemics cannot justify the aberrant serotype distribution. In addition, no seasonal distribution of pneumococcal serotypes was observed.
For its effects to be fully evident, replacement requires a long duration of vaccine use and high vaccination coverage.14 Several non-PCV13 serotypes were detected in our study population as causes of pneumococcal disease. This highlights the importance of strict monitoring of pneumococcal distribution after PCV introduction to evaluate the real protection offered by the available vaccines and the need for different protection from pneumococcal AOM.
We thank the Milan AOM Study Group (Paola Gallia, Elisabetta Mazzucchi, Josè Onorato, Annarita Russo, Anna Manuela Vaccarino) for the support in enrolling the cases and Calogero Sathya Sciarrabba for data entry.
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