Pneumococcal serotypes in children and relationship with clinical presentation and antimicrobial susceptibility in the PCV13 era

Background: The aim of this study was to analyse the serotypes causing invasive pneumococcal disease (IPD) according to the clinical presentation, and antimicrobial susceptibility in children aged ≤17 years before the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in the official paediatric schedule. Methods: We conducted a prospective study in children ≤17 years with IPD attended in three Catalan hospitals between January 2012 and June 2016. IPD cases were diagnosed by culture or RT-PCR. Demographic, microbiological, and clinical data were analysed. Associations were assessed using the odds ratio (OR) and 95% confidence intervals (CI). Results: Of the 253 cases included, 34.4% were aged < 2 years, 38.7% 2-4 years and 26.9% 5-17 years. Over 64% of cases were PCV13 serotypes. Cases with PCV13 serotypes were associated with overall pneumonia (OR:7.474.0-13.96) and complicated pneumonia (OR:7.24.04-12.75), overall and in each age group (p<0.05). Serotypes 3 and 1 were associated with complicated pneumonia (p<0.05). Non-PCV13 serotypes were associated with meningitis (OR:7.322.33-22. 99) and occult bacteremia (OR:3.61.56-8.76). Serotype 19A was more frequent in children aged <2 years and serotype 3 and 1 in those aged 2-4 years and >4 years, respectively. Forty-four cases (36.1%) were non-susceptible to penicillin and 16.4% were also non-susceptible to cefotaxime. There were no significant differences between PCV13 and non-PCV13 cases with non-susceptible penicillin strains (36.1% and 36.0%, respectively), while PCV13 cases showed more frequently non-susceptible cefotaxime strains (23.6%; p=0.010). Serotypes 19A and 14 were associated with non-susceptibility to both penicillin and cefotaxime

In Catalonia, Spain, PCV7 was available in 2001, but only children with risk factors were vaccinated for free [8,9]. An estimated vaccine coverage of around 50% was reached [9].Between 2005 and 2007, a change in IPD serotypes was found in children aged <2 years, compared with the pre-vaccine period, with a decrease in PCV7 serotypes and an increase in non-PCV7 serotypes [10]. PCV13 replaced PCV7 in 2010 and finally, in July 2016, was included in the paediatric official calendar [11].
PCV13 could cover up to 70% of IPD cases in our reference area [12].
Studies worldwide have found that the changing distribution of pneumococcal serotypes has led to changes in the rates of S. pneumoniae resistance to antibiotics [13,14].
The aim of this study was to analyse the distribution of S. pneumoniae serotypes according to the clinical presentation of IPD and antimicrobial susceptibility in children aged <18 years in a community with intermediate PCV13 coverage before the introduction of this vaccine in the official paediatric schedule.

Study design
We conducted a prospective observational study between January 2012 and June 2016 in children IPD was diagnosed through the isolation of S. pneumoniae by culture or detection of bacterial DNA by real-time polymerase chain reaction (RT-PCR) in any normally sterile site [15].

Data collected
Epidemiological and clinical characteristics, including age, gender, underlying medical condition, clinical presentation, intensive care unit (ICU) admission, clinical outcome and PCV13 vaccination status were collected. The method of diagnosis, serotype and antimicrobial susceptibility were also analysed.
A subject was considered vaccinated if they had received the last dose of PCV13 ≥15 days before symptom onset.

Serotype identification and antimicrobial susceptibility
Strains isolated by culture were serotyped using the Quellung reaction or dot-blot at the National Centre of Microbiology (Majadahonda, Spain) [16].When the diagnosis was made only by RT-PCR, serotypes were identified at the Molecular Microbiology Department, Hospital Sant Joan de Déu in accordance with previously-validated methods [15].The procedure allows the identification of 40 serotypes by a RT-PCR threshold cycle ≤30. Associations between variables were assessed using the odds ratio (OR) and 95% confidence intervals (CI).
The analyses were conducted using the Statistical Package for Social Sciences (SPSS 19.0 for Windows) and EPIDAT (program for the epidemiological analysis of tabulated data; version 3.1).

Data confidentiality and ethical aspects
Informed consent was obtained from all individual participants included in the study. The study complies with the principles of the Declaration of Helsinki and the legal structure with respect to international human rights and biomedicine and protection of personal data laws. The Ethics Committee of Hospital Sant Joan de Déu approved the study. Two patients died: an 18-month-old child with serotype-35F meningitis who had congenital immunodeficiency, and a 23-month-old child with septic shock caused by a non-vaccine serotype which was not identified.
There were no significant differences in the percentage of non-susceptible penicillin strains between PCV13 serotypes (36.1%) and non-PCV13 serotypes (36.0%). PCV13 serotypes were associated with strains not susceptible to cefotaxime (p = 0.010) and with isolates not susceptible to both penicillin and cefotaxime (p = 0.010).
Serotype 11A showed non-susceptibility to cefotaxime in 60% of strains compared with 14.5% for other serotypes (p = 0.031). These strains also were non-susceptible to penicillin (p = 0.031).
Other serotypes showed significant differences in antimicrobial susceptibility, namely 24F which showed penicillin MIC >0.06 mg/L in 83.3% (p = 0.023), and 23B in 100% (p = 0.045). Among the strengths of the study is the high percentage of cases serotyped, since serotyping was not possible in only 3.8% of cases. In addition, the same diagnostic techniques (culture and PCR) were used uniformly in all cases, improving both the diagnostic sensitivity and the range of serotypes identified, which permitted a more precise estimate of the burden of IPD.

Discussion
There were some limitations. First, the population studied corresponds to three paediatric hospitals, although these hospitals serve 32% of the population aged <18 years requiring hospitalization in Catalonia. In the subgroup analyses, the small number of cases observed in some situations may explain that the confidence intervals of the associations were very wide. Finally, antibiotic sensitivity could only be studied in 48% of cases serotyped, since a large part of the cases were diagnosed by PCR.

Conclusions
PCV13 serotypes were the most frequently found IPD serotypes in children aged <18 years, especially serotype 19A in children aged <2 years, serotype 3 in the 2-4 years age group and serotype 1 in the 5-17 years age group.
Non-PCV13 serotypes were the main cause of meningitis, occult bacteraemia and other clinical presentations, while PCV13 serotypes were mainly responsible for pneumonia.
PCV13 and non-PCV13 cases presented high frequency of non-susceptibility to penicillin. Nonsusceptibility to both penicillin and cefotaxime was associated with serotypes 19A and 14, and serotype 19A was associated with resistance to penicillin.
The non-susceptibility to antibiotics of non-PCV13 serotypes is a worrisome fact and should be monitored to apply the appropriate disease prevention strategies.

Consent for publication
Not applicable

Availability of data and material
The dataset supporting the conclusions of this article is included within the article.

Competing interests
JJ Garcia-Garcia has received honoraria for speaking at symposia (Pfizer and GSK), and Financial support for attending symposia (Pfizer). All outside the submitted work.
C Muñoz-Almagro reports travel grants from Pfizer, research grants from BioMerieux, Stat DX and Instituto de Salud Carlos III, personal fees from GSK as consultor advisor board and honoraria for speaking at symposium from Roche and Biomerieux. All outside the submitted work.
For de remaining author none were declared.

This work was supported by the Plan Nacional I+D+I, Instituto de Salud Carlos III -Subdirección
General de Evaluación y Fomento de la Investigación Sanitaria (Projects: PI11/02081 and PI11/02345) and cofounded by the Fondo Europeo de Desarrollo Regional (FEDER) (their funding role was for serotyping study and for participating in congresses) and the Agència de gestió d' ajuts universitaris i recerca (AGAUR: grant number 2017 SGR 1342) (its funding role was for English manuscript revision).    "ns" indicates p value≥0.05.