Journal of Medical Microbiology

Journal of Medical Microbiology Genetic profiles and antimicrobial resistance of Streptococcus pneumoniae non-PCV10 serotype isolates recovered from meningitis cases in Salvador, Brazil --Manuscript Draft-Manuscript Number:

JMM-D-16-00266R1

Full Title:

Genetic profiles and antimicrobial resistance of Streptococcus pneumoniae nonPCV10 serotype isolates recovered from meningitis cases in Salvador, Brazil

Short Title:

S. pneumoniae non-PCV10 serotypes from meningitis cases

Article Type:

Standard

Section/Category:

Microbial Epidemiology

Corresponding Author:

Leila Carvalho Campos, Ph.D. Centro de Pesquisas Goncalo Moniz Salvador, Bahia BRAZIL

First Author:

Jailton Azevedo, Ph.D.

Order of Authors:

Jailton Azevedo, Ph.D.

Downloaded from www.microbiologyresearch.org by IP: 158.111.236.10 On: Mon, 12 Sep 2016 20:18:37

Eder Silva dos Anjos, M.Sc. Soraia Machado Cordeiro, Ph.D. Milena Soares dos Santos, Ph.D. Eliane Cunegundes Escobar, B.Sc. Paulo Rocha Lobo, M.D. Maria da Gloria Carvalho, Ph.D. Mitermayer Galvão Reis, Ph.D., M.D. Joice Neves Reis, Ph.D. Leila Carvalho Campos, Ph.D. Abstract:

In 2010, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced to the Brazilian childhood vaccination program. Concerns have been raised that nonvaccine serotypes could increase in prevalence and reduce the benefits of vaccination; therefore, we examined the non-PCV10 isolates recovered from meningitis during pre (January, 2008-May, 2010) and post-vaccine (June, 2010-December, 2012) periods. Surveillance for pneumococcal meningitis was established at the Reference Hospital of Infectious Diseases in Salvador, Brazil. Serotypes were determined by multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility testing was conducted by E-test and broth microdilution. Genotyping employed pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 148 cases of meningitis were identified from January 2008 to December 2012, 77 (52%) of which were due to nonPCV10 isolates, with 50 (52.1%) from pre-vaccine and 27 (52%) post-vaccine periods. In the post-vaccine period, the non-PCV10 serotypes 12F (n = 6; 22.2%), 10A (n = 3; 11.1%), 15B (n = 2; 7.4%), and 18B (n = 2; 7.4%) were the most prevalent. Forty-three isolates (55.8%) were non-susceptible to one or more antibiotics. Non-susceptibility to penicillin was observed among serotypes 19A (3 isolates), 9N (1 isolate), and 12F (1 isolate). PFGE and MLST results demonstrated a wide genetic diversity among the isolates. During the early period following PCV10 introduction, no obvious emergence of a particular serotype was evident among non-PCV10 strains. This study underscores the importance of monitoring any changes among non-PCV10 cases after the introduction of PCV10.

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Genetic profiles and antimicrobial resistance of Streptococcus pneumoniae non-PCV10

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serotypes isolates recovered from meningitis cases in Salvador, Brazil

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Running Title: S. pneumoniae non-PCV10 serotypes from meningitis cases

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Jailton Azevedo1, Éder Silva dos Anjos1, Soraia M. Cordeiro2, Milena S. dos Santos3,

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Eliane C. Escobar1, Paulo R. Lobo1, Maria da Glória Carvalho4, Mitermayer G. Reis1,

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Joice N. Reis1, 2, Leila C. Campos1*


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Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, 40296-710, Brazil

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Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, Bahia, 40170-115,

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Brazil

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Bahia, Vitória da Conquista, Bahia, 45029-094, Brazil

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30333, USA

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Keywords: Streptococcus pneumoniae, genotype, PCV10, surveillance.

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Subject Category: Microbial Epidemiology

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Word Count: 3089

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*Corresponding author: Laboratório de Patologia e Biologia Molecular, Instituto

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Gonçalo Moniz, Fundação Oswaldo Cruz, 40296-710 Salvador, BA, Brazil. Tel.: +55

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71 31762350. E-mail address: [email protected] (L. C. Campos)

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The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Instituto Multidisciplinar em Saúde, Campus Anísio Teixeira, Universidade Federal da

Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta,


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ABSTRACT

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In 2010, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced to the

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Brazilian childhood vaccination program. Concerns have been raised that non-vaccine

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serotypes could increase in prevalence and reduce the benefits of vaccination; therefore,

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we examined the non-PCV10 isolates recovered from meningitis during pre (January,

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2008–May, 2010) and post-vaccine (June, 2010–December, 2012) periods. Surveillance

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for pneumococcal meningitis was established at the Reference Hospital of Infectious

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Diseases in Salvador, Brazil. Serotypes were determined by multiplex PCR and/or

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Quellung reaction. Antimicrobial susceptibility testing was conducted by E-test and

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broth microdilution. Genotyping employed pulsed field gel electrophoresis (PFGE) and

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multilocus sequence typing (MLST). A total of 148 cases of meningitis were identified

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from January 2008 to December 2012, 77 (52%) of which were due to non-PCV10

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isolates, with 50 (52.1%) from pre-vaccine and 27 (52%) post-vaccine periods. In the

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post-vaccine period, the non-PCV10 serotypes 12F (n = 6; 22.2%), 10A (n = 3; 11.1%),

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15B (n = 2; 7.4%), and 18B (n = 2; 7.4%) were the most prevalent. Forty-three isolates

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(55.8%) were non-susceptible to one or more antibiotics. Non-susceptibility to

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penicillin was observed among serotypes 19A (3 isolates), 9N (1 isolate), and 12F (1

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isolate). PFGE and MLST results demonstrated a wide genetic diversity among the

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isolates. During the early period following PCV10 introduction, no obvious emergence

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of a particular serotype was evident among non-PCV10 strains. This study underscores

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the importance of monitoring any changes among non-PCV10 cases after the

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introduction of PCV10.

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INTRODUCTION

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Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide,

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causing diseases that range in severity from meningitis, septicaemia, and pneumonia, to

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sinusitis and acute otitis media (WHO, 2012). Almost all isolates that cause these

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infections are encapsulated, and to date, greater than 94 different pneumococcal

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serotypes have been distinguished, expressing structurally and antigenically different

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capsular polysaccharides (Geno et al., 2015). The capsule is the target of all licensed

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vaccines (Feldman & Anderson, 2014).


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The licensure and subsequent widespread use of pneumococcal conjugate vaccines has

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decreased the overall incidence of invasive pneumococcal disease in many countries, in

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part by also reducing carriage of vaccine-type strains, and inducing herd immunity

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(Millar et al., 2008; Hammitt et al. 2014). However, concerns have been raised that non-

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vaccine serotypes could increase in prevalence and reduce the benefits of vaccination

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(Pilishvili et al., 2010).

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The overall effect of the use of conjugate vaccines can include serotype replacement

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and/or the capsular switching phenomenon. The contribution of each of these effects in

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geographical areas where a conjugate vaccine is introduced is difficult to predict;

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therefore, careful monitoring of the epidemiology and understanding of the dynamics of

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the pneumococcal population is required to assess the initial and long-term benefits of

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vaccination in each region. In 2010, Brazil introduced the 10-valent non-typable

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Haemophilus influenzae protein D conjugate vaccine (PCV10 or PHiD-CV; GSK

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Biologicals, Rixensart, Belgium) into its routine National Immunization Program, in a

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three-dose scheme (2, 4, and 6 months) plus a booster at 12 months. This vaccine

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contains antigens designed to protect against the serotypes 4, 9V, 14, 19F, 23F, 18C,

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and 6B, in addition to serotypes 1, 5, and 7F (Prymula & Schuerman, 2009).

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Since 1996, hospital-based surveillance for pneumococcal antibiotic resistance and

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capsular serotypes associated with pneumococcal meningitis has been conducted in

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Salvador, Bahia (de O. Menezes et al., 2011; dos Santos et al., 2015; Leite et al., 2016).

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Here we describe the genetic diversity and antimicrobial susceptibility of S. pneumoniae

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non-PCV10 serotype isolates recovered from patients with meningitis before (January

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2008–May 2010) and after (June 2010–December 2012) the introduction of PCV10 in

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Salvador, Bahia.

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METHODS

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Study population and surveillance system. In 1996, an active surveillance system for

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pneumococcal meningitis cases was initiated at the Hospital Couto Maia, a reference

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public hospital for infectious diseases with 120 beds, which functions as the medical

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attendance centre for meningitis in the metropolitan region of the Salvador municipality

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(3,573,973 inhabitants, 2010 IBGE census). The state health department requires all

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suspected meningitis cases from the region to attend this hospital, and more than 90% of

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meningitis reports from the region are reported at this unit (DATASUS, 2015). Our

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technicians reviewed the daily clinical laboratory records at the hospital to identify all S.

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pneumoniae isolates obtained from symptomatic patients with meningitis with positive

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cerebrospinal fluid (CSF) and fulfilling the criteria for bacterial meningitis (≥ 100

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leukocytes/mm3). The pneumococcal isolates identified in this hospital were sent to the

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Instituto Gonçalo Moniz, Salvador, Brazil for confirmation.


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Microbiological procedures. Pneumococcal strains were identified by standard

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methods, including Gram stain, colony morphology on agar media with 5% sheep

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blood, optochin susceptibility (5-µg Oxoid disks) and bile solubility (Werno &

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Murdoch, 2008). Capsular serotypes were determined by multiplex-PCR (Dias et al.,

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2007; CDC, 2014). Quellung reaction tests were performed by the Streptococcus

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Laboratory at CDC, Atlanta, for all serotypes not resolved by PCR assay, as well as for

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10% of the serotypes resolved by PCR, as a quality control measure. Antimicrobial

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susceptibility to penicillin, cefotaxime, clindamycin, chloramphenicol, erythromycin,

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levofloxacin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin (Sigma-

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Aldrich, Germany) was assessed by broth microdilution according to Clinical and

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Laboratory Standards Institute (CLSI) guidelines (CLSI, 2014). The reference strain S.

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pneumoniae ATCC 49619 was used for quality control. All isolates with minimal

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inhibitory concentration (MIC) values ≥ 0.12 µg/mL were defined as pneumococcal

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non-susceptible to penicillin.

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For comparison purposes, non-PCV10 serotypes were separated into two periods: 1)

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Pre-vaccine period (January 2008 to June 2010), with 50 (52.1%) isolates, and 2) post-

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vaccine (July 2010 to December 2012), with 27 (52%) isolates.

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Molecular characterisation. Non-PCV10 isolates were characterised by pulsed-field

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gel electrophoresis (PFGE). Chromosomal digests generated by SmaI were prepared and

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analysed as described elsewhere (McEllistrem et al., 2000). Fragments were separated

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by PFGE in 2% agarose gels using CHEF-DRII apparatus (Bio-Rad Laboratories,


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Hercules, CA) with pulse times of 2 to 30 s for 19 h at 14°C and 6 V/cm. Restriction

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profiles were analysed using the GelCompar II software package (version 4.0; Applied

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Maths, Bionumerics) to compare the band patterns. PFGE patterns were clustered by the

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unweighted-pair group method using average linkages (UPGMA), and a dendrogram

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was generated from a similarity matrix calculated using the Dice similarity coefficient,

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with an optimization of 1.0% and a tolerance of 1.5%. PFGE patterns were defined as

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isolates with a similarity of ≥ 80% in the dendrogram. According to PFGE clustering

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analysis, based on similarity profiles, a random sample of all of isolates showing high

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relatedness (≥ 80%) from the pre- (26/50 isolates) and post- (14/27 isolates) PCV10

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periods were selected for MLST analysis, according to an adaption of the method

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described by Enright & Spratt (1998) (detailed at

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http://www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm).

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Data analysis. Epidemiological and laboratory information were entered into a standard

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database and analysed using Epi-Info Version 3.5.1 (CDC, Atlanta, GA). We calculated

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annual incidence rates (cases per 100,000 population) by dividing the number of cases

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among residents of the Salvador municipality by the estimated population, using 2010

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census bureau data to calculate rates for 2008 through 2012 (IBGE, 2010). Fisher’s

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exact or chi-square tests were used to compare differences between proportions for

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dichotomous variables. All p values were based on two-sided tests, and p < 0.05 was

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considered statistically significant.

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Ethical approval: Informed consent procedures were applied to all patients and/or

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guardians of patients included in this study, which was approved by the National

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Committee for Research Ethics (CONEP) and the FIOCRUZ Institutional Review

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Board, Brazilian Ministry of Health (no. 044/2013). All patients or legal guardians gave

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written informed consent prior to enrolment of subjects in the study.

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RESULTS

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Overall patient characteristics


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A total of 148 cases of pneumococcal meningitis were identified from January 2008 to

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December 2012, 77/148 (52%) of which were due to non-PCV10 isolates, with 50/96

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(52.1%) from pre-vaccine and 27/52 (52%) from post-vaccine periods (Table 1). Total

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non-PCV10 cases consisted of 32.5% women and 67.5% men (Table 1). Of the 71

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patients for whom age information was available, 8 (16.5%) were children aged ≤ 5

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years that occurred only in the pre-PCV10 period (p = 0.04). The majority of cases were

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diagnosed among adults aged between 19–49 years (41.7% and 60.9% from the pre- and

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post-vaccine periods, respectively) and ≥ 50 years (22.9% and 26.1%, from the pre- and

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post-vaccine periods, respectively). Differences in the number of days of hospitalization

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(14 vs. 22; p = 0.04) and in the CSF protein content (300 md/dL vs. 500 md/dL; p =

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0.03) were observed between the pre- and the post-vaccine periods. No differences were

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observed in respect of antibiotic resistance or acute illness preceding meningitis.

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The annual incidence of pneumococcal meningitis (PCV10 and non-PCV10 cases)

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decreased from 0.9/100,000 inhabitants (30 cases) in 2008 to 0.36/100,000 (12 cases)

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inhabitants in 2012 (p < 0.05). Fig. 1 shows the annual incidence of pneumococcal

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meningitis stratified by PCV10 and non-PCV10 cases during the study period. In

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general, there was a decrease in the incidence of both PCV10 and non-PCV10 cases

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during the study period. In particular, we observed a decrease in the incidence of non-

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PCV10 cases from 0.69/100,000 inhabitants in 2008 to 0.21/100,000 inhabitants in 2012

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(p < 0.76). For PCV10 cases, the incidence reduced from 0.57/100,000 inhabitants in

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2010 to 0.21/100,000 inhabitants in 2012 (p < 1.0) after the introduction of the PCV10

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vaccine in June 2010.

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Serotype distribution and antimicrobial susceptibility

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Over the study period, 28 different serotypes were found: six occurred only in the post-

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vaccine period (10F, 11A, 21, 22F, 23B, and 24F), whereas ten serotypes (6A, 6C, 17F,

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18A, 19A, 20, 23A, 28A, 35F, and 38) were identified only in the pre-vaccine period.

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Twelve serotypes were found in both periods (3, 7C, 8, 9N, 10A, 12F, 13, 15B, 16F,

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18B, 34, and 35B). In the pre-vaccine period, the most frequent non-PCV10 serotypes

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were as follows: 3 (n = 6, 12%), 19A (n = 4, 8%), and 6A (n = 4, 8%). In the post-

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vaccine period, non-PCV10 serotypes 12F (n = 6, 22.2%), 10A (n = 3, 11.1%), 15B (n =

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2, 7.4%), and 18B (n = 2, 7.4%) were the most prevalent. A slight increase in the

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number of 12F serotype cases was detected in the post-vaccine period (Fig. 2).

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Of 77 non-PCV10 isolates characterised, 43 (55.8%) were non-susceptible to one or

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more antibiotics. All isolates were susceptible to cefotaxime, clindamycin,

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chloramphenicol, levofloxacin, and vancomycin. A total of 28 (36.4%) and 16 (20.8%)

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isolates were resistant to trimethoprim-sulfamethoxazole and tetracycline, respectively.

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Serotypes 19A (3 isolates), 9N (1 isolate), and 12F (1 isolate) were not susceptible to

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penicillin. One isolate (serotype 21) from the post-vaccine period was erythromycin-

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resistant (Fig. 3).


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PFGE and MLST analysis

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We identified 45 isolates grouped into 19 PFGE profiles with ≥ 80% similarity. Among

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those profiles, 14/19 (73.7%) and 5/19 (26.3%) occurred in the pre- and post-vaccine

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periods, respectively. Overall, 32/77 (41.5%) of isolates were not clustered due to

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exhibiting < 80% similarity in PFGE, with higher variability in the post-vaccine period

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(17/27; 63%). Sequence types (STs) 80 (n = 3 isolates), 193 (n = 2 isolates), and 8376

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(n = 2 isolates; serotype 12F) were found in both pre- and post-vaccine periods. The two

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12F isolates (ST8376) were susceptible and resistant to penicillin in the pre-vaccine and

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post-vaccine periods, respectively. Seven STs were newly assigned in our study, of

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which five were from the pre-vaccine period (ST9678, ST9679, ST9680, ST9070, and

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ST9071), one from the post-vaccine period (ST9681), and was one detected in both

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periods (ST8376). Furthermore, in the pre-vaccine period, two serotypes (3 and 35B)

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were found to belong to the same ST (ST180), while in the post-vaccine period,

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serotypes 12F and 21 (resistant to STX and ERI) showed identical PFGE profile and the

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same ST (ST218).

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DISCUSSION

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In Brazil, the introduction of PCV10 to the childhood immunisation schedule in 2010

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resulted in several changes in the epidemiology of cases of pneumococcal meningitis; in

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particular, we observed an overall decrease in the number of cases. During the years


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following its introduction, vaccination coverage rates improved dramatically, with

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38,035 doses applied in 2011 (73% of the target population) and 42,415 doses applied

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in 2012 (81.5% of the target population)—levels which are considered satisfactory

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(DATASUS, 2015).

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We did not observe any post-PCV10 emergence of a particular serotype in this study.

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The PCV10 vaccine was only recently introduced to our region, and 2010 was a year of

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transition. Furthermore, comparison of the number of 12F, 10A, 15B, and 18B serotype

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isolates identified in the post-vaccine period with those obtained in past years indicated

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a normal fluctuation of these serotypes over the years, with no evidence of specific

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emergence of any particular serotype (data not shown). Fluctuations in the prevalence of

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a particular serotype may occur naturally in the population of pneumococci, even in the

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absence of the selective pressure imposed by the use of conjugate vaccines (Finland &

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Barnes, 1977; Lagos et al., 2008; Ruckinger et al., 2008). Continued surveillance over a

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longer period is needed to fully assess the impact of vaccine introduction in our region

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on the pneumococci population.

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In this study, only four isolates of serotype 19A were identified in the pre-vaccine

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period. These findings are in agreement with those of dos Santos et al. (2013) in São

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Paulo, which did not show an increased incidence of this serotype in that city during the

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post-vaccine period (dos Santos et al., 2013). The incidence of serotype 19A has

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remained low and stable in Latin America and the Caribbean for 20 years (Castaneda et

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al., 2012), and a recent study in Brazil demonstrated the specific effectiveness of

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PCV10 against serotype 19A (Domingues et al., 2014). Regarding antimicrobial

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susceptibility, three of four serotype 19A isolates identified in the pre-vaccine period

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were penicillin-non-susceptible. In the United States, after PCV7 vaccination, serotype

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19A emerged as an important cause of invasive pneumococcal disease, and the most

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common 19A clones were associated with increased multi-resistance to penicillin and

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other antimicrobials (Pilishvili et al., 2010).

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Studies of the immunogenicity of the PCV10 vaccine show that, although it is active

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against serotype 6A, its activity is approximately 33%–50% lower than that observed

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with PCV7. Therefore, it is not yet clear whether PCV10 induces cross-protection

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against serotype 6A (Vesikari et al., 2011; Farkouh et al., 2012). In this study, serotype

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6A was found only in the pre-vaccine period.


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Of the 77 isolates characterised in this study, 43 (55.8%) exhibited resistance to at least

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one antimicrobial, and 13.5% of the isolates (n = 7) showed resistance to two or more

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antibiotics. Multidrug resistance to antibiotics in S. pneumoniae is undoubtedly a cause

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for concern. However, in the United States, the introduction of PCV13 in 2010 led to a

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reduction in resistant infections (Moore et al., 2015).

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The evidence of non-susceptibility to penicillin in the single serotype 12F isolate of the

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post-vaccination period suggests that this serotype should be followed in the

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epidemiological surveillance system in our region, especially if this lineage becomes

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established as the most prevalent over time. A prospective study with a longer period of

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monitoring may provide conclusive information. The resistance to trimethoprim-

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sulfamethoxazole (39%) and tetracycline (20.8%) observed in this study is consistent

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with previous work in our region (de O. Menezes et al., 2011), and with another

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national study (Brandileone et al., 2006) and, may be partly associated with the previous

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use of antimicrobials. We found resistance to erythromycin in only one isolate (serotype

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21) of the post-vaccine period, in contrast to observations elsewhere; in fact, in the

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U.S.A. macrolide resistance is overtaking penicillin-resistance, due to the removal of

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PCV13 strains (especially 19A), leaving serotypes like 33F which are primarily

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macrolide-resistant (Metcalf et al., 2015).

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Genotypic characterisation of the S. pneumoniae isolates by PFGE showed great genetic

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diversity among the non-PCV10 serotypes in the post-vaccine period (63%). In the pre-

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vaccine period, one 9N isolate was identified as belonging to ST66. It is important to

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note that this ST, associated with serotype 14, is considered one of the leading and most

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prevalent clones associated with invasive disease in Salvador and other regions of

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Brazil (Brandileone et al., 1997; Reis et al., 2008). ST180 (serotype 3), ST193 (serotype

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18B), and ST218 (serotype 12F) identified in our study are related to the globally

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disseminated clones, Netherlands3-31, Greece21-30, and Denmark12F-34, respectively, as

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described by the Pneumococcal Molecular Epidemiology Network (PMEN)

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(http://web1.sph.emory.edu/PMEN/). This molecular surveillance also encountered

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similar PFGE profiles (relatedness ≥ 80%) exhibiting different capsular serotypes,

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suggesting the occurrence of capsular switching events in this epidemiological setting.

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However, this finding requires careful interpretation and further investigation using a

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more complete molecular approach.


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As pointed out by Afonso et al. (2013), any study that uses a temporal method to

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determine the initial effects of a vaccine can be subject to variation depending on

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vaccination coverage and the natural lag period between the start of a vaccination

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program and protection in a population (Afonso et al., 2013). This study was conducted

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in the pre- and early post-implementation stages of the PCV10 vaccine program in a

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large urban centre and any conclusions that can be drawn from the results obtained here

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should be considered preliminary observations of the distribution and molecular

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characteristics of non-PCV10 serotypes in our region in the post-vaccine period. The

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scenario that we describe here could change as the use of the PCV10 vaccine is

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expanded and a larger number of isolates can be studied. The limitations of this study

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include the small number of isolates and the fact that the data may have been collected

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too early in the vaccination program to detect replacement strains. Furthermore, the

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majority of cases were diagnosed in adults (63.4%), a group to which the vaccine does

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not apply. The effects of the eventual replacement of PCV10 would need an even longer

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period to be detected.

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It is important to note the evidence of an isolate from the pre-vaccine period expressing

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capsular type 35B and sequence type 180, which had not been described before in the

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MLST database (http://spneumoniae.mlst.net/). Similarly, the ST 218 has never

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previously been identified in serotype 21 (resistant to STX and ERI), which had the

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same PFGE profile as serotype 12F in our study.

293 294

In conclusion, our study demonstrates that, during the early period following PCV10

295

introduction, no obvious emergence of a particular serotype was evident among non-

296

PCV10 strains. Further studies are necessary to confirm the occurrence of capsular

297

switch events in those serotypes presenting the same PFGE profile and the same ST.

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The results of our study provide the basis for future analyses regarding the effects of

299

vaccination in the years following PCV10 introduction and for monitoring changes in

300

population biology associated with vaccine introduction.

301 302

ACKNOWLEDGEMENTS

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We thank Ana Paula de Oliveira Menezes for her help with PCR assays. We are also

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grateful to Silvana Paz for her help in providing sequencing data and to Bernard Beall

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(Centers for Disease Control and Prevention) for full revision of the manuscript. This

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work was supported by the Conselho Nacional de Desenvolvimento Científico e

307

Tecnológico (483674/2010-9) and Fundação Oswaldo Cruz (PAPES 407551/2012-3).

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ABBREVIATIONS

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CLSI, Clinical and Laboratory Standards Institute; MLST, multi-locus sequence

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typing; Non-PCV10, non-vaccine type; PCV10, 10-valent pneumococcal conjugate

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vaccine; PFGE, pulsed field gel electrophoresis; ST, sequence type.


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REFERENCES

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454

Figure Titles

455 456 457

Fig. 1. Incidence and number of cases of pneumococcal meningitis in the metropolitan region of Salvador, Brazil, stratified by PCV10 and non-PCV10 serotypes, 2008–2012.

458 459

Fig. 2. Distribution of non-vaccine serotypes among isolates from pneumococcal

460

meningitis cases in the metropolitan area of Salvador, 2010–2012, stratified by pre-

461

vaccine and post-vaccine periods (n = 77).

462 463

Fig. 3. Dendrogram of PFGE molecular profiles of non-PVC10 serotypes isolated from

464

pneumococcal meningitis in the metropolitan area of Salvador. A: Pre-vaccine period (n

465

= 50); B: Post-vaccine period (n = 27).

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466

Table 1. Characteristics of patients with pneumococcal meningitis caused by non-PCV10 pneumococci before and after vaccination.


Characteristics Demographic data Male gender Age groups (years), median (range) ≤5 6–18 19–49 ≥ 50 Clinical information Neurological status at admission* Days of symptoms, median (range) Days of hospitalisation, median (range) ICU admission (days), median (range) Mortality CSF information White cell count (cells/mm3), median (range) Protein (mg/dL), median (range) Glucose (mg/dL), median (range) Cases non-susceptible to: Penicillin Tetracycline Trimethoprim-sulfamethoxazole Erythromycin Acute illness preceding meningitis (%) Pneumonia Acute otitis media Head trauma Upper respiratory tract infection

Pre-PCV10 period n = 50 (100%)

Post-PCV10 period n = 27 (100%)

467

p-value

34 (68%) 26 (10.5–46.5) 8 (16.5%) 9 (18.8%) 20 (41.7%) 11 (22.9%)

18 (69%) 36 (27–53) 0 (0) 3 (13%) 14 (60.9%) 6 (26.1%)

1.00 0.07 0.04 0.58 0.14 0.79

33 (66%) 3 (1–4.5) 14 (11–20) 13 (6–24) 18%

18 (69%) 2 (1–3) 22 (15–32) 10 (7–21) 15%

0.96 0.29 0.03 0.78 0.97

4150 (627–10000) 300 (300 – 500) 20 (20–40)

3840 (1100–10000)

0.91

500 (350–565) 20 (20–32)

0.03 0.60

4 (8%) 13 (26%) 19 (38%) 0 (0)

1 (3.7%) 3 (11.1%) 11 (47.7%) 1 (3.7%)

0.53 0.13 0.82 0.35

6 (12%) 4 (8%) 6 (12%) 12 (24%)

0 (0) 2 (7.7%) 1 (3.9%) 3 (11.5%)

0.65 0.65 1.00 0.19

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499


16

500 *coma or altered mental status; ICU: Intensive care unit (21 patients were admitted).

Figure 1

Click here to download Figure Fig 1.docx

0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

35 30 25 20 15 10 5 0


2008

2009 2010 2011 Study Year

PCV10 serotypes*

2012

Non-PCV10 serotypes

Numer of cases, overall

Incidence (cases/100,000 inhabitants)

Fig. 1


Number of cases

Figure 2 Click here to download Figure Fig 2.docx

Fig. 2

Pre-PCV10 Period

Serotypes

Post-PCV10 Period

Figure 3

Click here to download Figure Fig. 3.docx

Fig. 3 (a)

(b)

Sample Serotype MLST

Resistance


profile

ST, sequence type; PEN, penicillin; TET, tetracycline; ERI, erythromycin; SXT, trimethoprim-sulfamethoxazole.

Sample Serotype MLST

Resistance profile