A 20-months Surveillance of Non-invasive Streptococcus Pneumoniae Infections in Belgium to Evaluate National Vaccination Strategy
Conditions: Pneumonia, Otitis Media, Sinusitis Bacterial
Sponsor: Sciensano

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A 20-months surveillance of non-invasive Streptococcus 
pneumoniae infections in Belgium  
to evaluate national vaccination strategy  
 
Protocol Version 1.2 
05/12/2019 
 

2 
 
 
0 Summary 
Population 
3,600 Belgian patients with community acquired pneumonia or acute otitis 
media, leading to positive S. pneumoniae from non-sterile clinical 
specimens. 
Intervention 
N/A 
Comparison 
Serotype distribution according to vaccination programs (campaigns) 
Outcome 
Primary: to establish insight in serotype dynamics for non-invasive 
pneumococcal diseases in Belgium (prospectively and retrospectively), in 
association with changing vaccination programs (campaigns). 
Secondary: surveillance of emerging serotypes, clones and drug 
resistances. Study of the genetic divergences between invasive and non-
invasive pneumococcal diseases isolates. 
Study Type 
Observational 
 
 
 
 
 
 
 
 
 
 
 
Principle Investigator 
Pieter-Jan Ceyssens, Ph.D 
Coordinating Scientist 
Ioannis Passaris, Ph.D 
Institute 
Sciensano 
Address 
J. Wytsmanstraat 14, 1050 Brussels, Belgium 
Contact 
+32 2 373 31 19                                      +32 2 373 33 23 
pieter-jan.ceyssens@sciensano.be         ioannis.passaris@sciensano.be 
 

3 
 
 
1 Study Rationale 
1.1 
Pneumococcal vaccination programs reduced burden of invasive disease 
Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide among 
children <2 years of age and in the elderly [1]. The pathogenicity of this bacteria can be divided into 2 
forms: invasive (IPD) and non-invasive pneumococcal disease (NIPD). Whereas the dangerous IPD 
type is responsible for meningitis and bacteraemia (septicaemia), the less severe NIPD can cause upper 
and lower respiratory tract infections such as community acquired pneumonia (CAP) and otitis media 
(OM). Of note, this subdivision between IPD and NIPD pneumococcal diseases is somewhat arbitrary 
since a substantial proportion (10-20%) of CAP pneumonia caused by S. pneumoniae is associated with 
contemporary bacteraemia (especially in elderly and debilitated patients). The distinction between PID 
and NIPD relates to the laboratory diagnosis and anatomical sites from which pneumococci are isolated. 
In PID, S. pneumoniae is detected from normally sterile body fluids (blood, cerebrospinal fluid (CSF), 
pleural, peritoneal or articular fluids). In NIPD, pneumococcal isolates are typically isolated from 
superficial body sites such as ear, nose, sputum or other lower respiratory tract sites (bronchial, 
endotracheal aspirates, bronchoalveolar lavage,...). There are over 90 pneumococcal serotypes, 
classified by an immunologically distinct capsule encoded by the capsular polysaccharide (cps) 
biosynthesis locus of the genome. The capsule is a major virulence factor and the basis for the currently 
licensed vaccines targeted toward the paediatric and elderly communities.  
In the course of the decades, various 
pneumococcal 
conjugate 
vaccines 
were 
successively introduced, with the 7-valent PCV7 
vaccine gradually being replaced by the 10-valent 
PCV10 and the 13-valent PCV13 [4]. These 
vaccination programs have led to drastic changes 
in the global epidemiology of pneumococcal 
diseases. 
Spawned 
surveillance 
data 
unequivocally showed strong decreases in IPD 
rates 
caused 
by 
the 
PCV 
serotypes. 
Simultaneously, serotype replacement by non-
vaccine 
targeted 
serotypes 
occupying 
nasopharyngeal niches vacated after vaccination 
was observed. A recent example of this is the 
emergence of multidrug resistant serotype 35B in IPD isolates from USA and France (Figure 1) [5-7].  
Further, an important, and as yet underestimated, role is thought to be played by non-encapsulated 
pneumococcal strains (NeSP), currently not targeted by vaccination. These strains have either deleted 
or dysfunctional cps genes, or have novel genes in place at this locus [8]. NeSP strains have been 
isolated from patients with invasive and non-invasive pneumococcal disease, but are typically more 
frequently associated with non-invasive diseases like conjunctivitis (85%) and OM (8%) [9].  
 
Figure 1. Number of serotype 35B invasive 
isolates by year from 1994 to 2014 in the USA, 
contrasting declining IPD incidence after vaccination 
campaigns. Figure from ref. [5]. 

4 
 
 
1.2 
Changing vaccination recommendations in Belgium 
In Belgium, the 23-valent polysaccharide vaccine or PPV23 (currently Pneumovax 23®, MSD) is 
licensed since 1995. It is currently recommended by the Superior Heath Council (CSS/HGR) for the 
prevention of pneumococcal infections in persons ≥2 years of age, presenting with an increased risk of 
mortality and severe morbidity due to pneumococcal infections, and often sequentially combined with 
PCV13 (Prevenar®, Pfizer) [4]. The national infant vaccination program was initiated in 2007 with PCV7 
(Prevenar®, Pfizer), and was updated to PCV13 in 2011. In 2015-2016, the program changed from 
PCV13 to PCV10 (Synflorix®, GSK). Very recently (HGR 9519, December 2018), a new advice was 
published by the Superior Heath Council which recommended again the use of the 13-valent vaccine in 
children <2 years of age, alongside an explicit statement stating that this advice could be reconsidered 
upon the release of new data. 
1.3 
The underdiagnosed and non-surveilled non-invasive population 
Current national and international pneumococcal surveillance is strongly focused on IPD. However, 
it is now commonly accepted that NIPDs by far account as the major part of all pneumococcal infections 
worldwide. O'Brien and colleagues reported in 2009 that out of 14.5 million pneumococcal cases, 95.6% 
were cases of NIPD [1]. In a more recent meta-study by the Johns Hopkins School of Medicine, it was 
estimated that for every adult bacteraemia case there are at least three non-invasive pneumococcal 
infections [10], resulting in huge societal and economic costs [11].  
Impelled by the lack of surveillance on NIPD, the majority of studies on pneumococcal vaccine 
effectiveness and serotype replacement are based on IPD data. However, sheer extrapolation of IPD 
dynamics to NIPD has been called into question by recent studies, using serotype-specific urinary 
antigen technology, which indicated discrepancy between persistent pneumococcal serotypes and the 
decline of the same serotypes causing IPD [12-14]. To get more insights in this matter, the Belgian 
Health Care Knowledge Centre (KCE) recently reviewed four observational studies from Denmark, 
Spain and Portugal (2), and the placebo arm of the CAPITA randomized controlled trial in the 
Netherlands [15-20]. In the infant populations of the study sites, PCV vaccine choice over time (PCV7, 
PCV10 and PCV13) and uptake differed. Three studies limited the detection of non-bacteraemic 
pneumonia to pneumonia with a positive culture of airway respiratory samples such as sputum or 
broncho-alveolar fluid, while the remaining study used both respiratory and urinary samples. Despite 
these differences, all studies concluded that the serotype distribution of bacteraemic and non-
bacteraemic pneumococcal disease differed substantially in terms of vaccine serotype coverage  
(Figure 2) [15].   

5 
 
 
 
 
 
 
These results concurred with the few non-EU studies conducted in USA, Britain and Korea, 
showing that frequencies of vaccine serotypes among NIPD vary between 28.2 and 57.4%, depending 
on the specimen type and patient age [21-23]. 
 
In Belgium, 78.4% of all IPD infections in 2017 were caused by non-vaccine targeted serotypes, 
which is more than what is found in our neighbouring countries. In infants below 2y, this figure drops to 
64.2% in 2018 (Figure 3). No information on serotype distributions for NIPD are available.  
1.4 
Sciensano holds historical collection of non-invasive pneumococcal isolates 
Between 1995 and 2015, Raymond Vanhoof (MD, PhD) led a team at Sciensano investigating the 
evolution of antibiotic resistance in NIPD isolates from CAP patients (as well as from children with acute 
otitits media). For 12 consecutive winter periods, his team collected between 300 and 500 clinical 
isolates using a network of 15 peripheral hospital-based laboratories distributed across the country. 
Consequently, we hold a collection of >5,000 strains for which extracted gDNA, complete phenotypic 
MIC profiles and associated metadata are already available (Figure 4 for age distribution). This study, 
funded by Bayer, focused on antimicrobial resistance and did not routinely included serotyping.  
 
Figure 
2. 
Proportion 
of 
serotype 
categories 
in 
bacteraemic/invasive 
pneumonia (BP) and non-
bacteriaemic/non-invasive 
pneumonia (NBP) in the 
Netherlands [16], Denmark 
[17], Spain [18] and Portugal 
[19,20]. 
Figure 
from 
the 
Belgian CSS/HGR rapport 
[15]. 
Figure 
3. 
Situation 
in 
Belgian 
infants 
(<2y). 
***serotypes present PCV13 
which are absent in PCV10. 
Data from the Belgian NRC 
for pneumococcal diseases.  

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This constitutes an extremely valuable collection that dates from before the introduction of 
pneumococcal vaccination programs. In the context of the proposed study, this collection would serve 
to retrospectively assess the impact of successive vaccination campaigns on serotype distribution 
among NIPD isolates in Belgium.  
 
 
1.5 
Implications for empiric treatment guidelines? 
Penicillin and macrolides have been the mainstay in the treatment of respiratory diseases for 
decades [25], but the worldwide spread of drug-resistant clones translated into increased usage of 
fluoroquinolones [26,27]. Recently, the World Health Organisation (WHO) included penicillin-resistant 
S. pneumoniae in the list of 12 critical drug-resistant pathogens [28]. Nevertheless, current empiric 
treatment guidelines support the continued use of penicillin and amoxicillin for drug-susceptible isolates. 
For risk patients, Belgian Antibiotic Policy Coordination Committee (BAPCOC) recommends use of 
either a respiratory fluoroquinolone, or combination therapy with beta-lactam antibiotic (high-dose 
amoxicillin, amoxicillin-clavulanate, or, alternatively, a second- or third-generation cephalosporin) plus 
a macrolide [29].  
Reminiscent of the studies on serotype replacement and global surveillance, studies on 
pneumococcal drug-resistance are strongly biased towards IPD isolates. However, it is conceivable that 
the non-invasive population functions as a ‘laboratory’ for the development of drug resistance. During a 
nation-wide multicentre survey spanning nearly two decades (1995-2014), researchers from Sciensano 
unequivocally reported higher rates of antibiotic resistance in the non-invasive population, as compared 
to the invasive strains (Figure 5) [30]. Therefore, including antimicrobial resistance screening for non-
invasive strains would improve pneumococcal surveillance, improve empiric treatment guidelines and 
add the possibility to detect circulating drug-resistant clones. 
 
Figure 4. Age distribution of the 
patients for which cultures were 
included during the NIPD surveillance 
study in 2012-2013 using the same 
network of laboratories [24]. 

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Figure 5.  Drug non-susceptibility in invasive versus non-invasive S. pneumoniae isolates in Belgium (2004-
2013). Data from IPD is annually reported by the National Reference Centre for pneumococcal disease. Data for 
NIPD was collected in the framework of the multicentre study (1995-2014) on drug susceptibility of non-invasive 
S. pneumoniae.  
1.6 
Key points  
From the information above, it is clear that: 
 
Current recommendations for vaccination and empiric treatment are strongly influenced by the 
current surveillance focus on IPD. Information on serotype distribution and drug resistance 
dynamics in NIPD is scarce. However, a discrepancy in serotype distribution between IPD and 
NIPD isolates is present in neighbouring countries.  
 
In Belgium, there was a recent (2015-2016) switch in the infant vaccination program to PCV10 
and a recommended reversal to PCV13 in December 2018. The influence on serotype 
distributions in NIPD is unclear, although this information is essential in vaccine cost-
effectiveness analyses [31]. 
2 Study aims 
The main objective of this study is to generate insight in the serotype dynamics for NIPD in Belgium, 
in association with changing vaccination programs. We aim to serotype non-invasive S. pneumoniae 
isolates prospectively collected in Belgian routine laboratories, during a period of 20 months. 
Simultaneously, a similar characterisation study will be performed retrospectively on a selection of more 
than 5,000 NIPD strains collected between 1995 and 2014 in Belgium. 
Additionally, this projects aims to do the surveillance of emerging serotypes, clones and drug 
resistances. Using Whole Genome Sequencing (WGS), the genetic divergences between IPD and NIPD 
isolates will also be studied, in collaboration with the National Reference Centre (NRC) for 
pneumococcal diseases (UZ Leuven, Belgium). 
3 Type of study 
This will be an observational study collecting data on S. pneumoniae serotypes causing NIPD in 
Belgium, in relation with changing vaccination programs. 
 
% resistant strains

8 
 
 
4 Conducting the study  
The study will be conducted by Sciensano in collaboration with the NRC for pneumococcal diseases 
and 12 peripheral clinical laboratories, which all agreed in writing to prospectively collect non-invasive 
S. pneumoniae samples in the framework of this study. These labs are geographically spread over 
Belgium to avoid clonal bias in sampling (Table 1).  
 
Table 1. Peripheral network of participating clinical laboratories. 
 Hospital 
 Contact 
 City 
  
 CHR Citadelle 
 Dr. M. Carpentier  Liège 
 
 CHU Mont-Godinne 
 Dr. D. Huang 
 Yvoir 
 ZNA 
 Dr. K. Camps 
 Antwerpen 
 Virga-Jessaziekenhuis  Dr. K. Magerman  Hasselt 
 Hôpital de Jolimont 
 Dr. N. Ciupilan 
 Haine-St.-Paul 
 AZ Delta  
 Dr. S. Vervaeke 
 Roeselare 
 AZ St. Jan 
 Dr. E. Nulens 
 Brugge 
 AZ. Jan Palfijn 
 Dr. L. Ide 
 Gent 
 Imeldaziekenhuis 
 Dr. J. Frans 
 Bonheiden 
 Hôpital Princesse Paola  Dr. Ph. Lefèvre 
 Marche-en-Famenne 
 Clinique St. Joseph 
 Dr. J.S. Goffinet 
 Arlon 
 CHU de Charleroi 
 Dr. S. Lali 
 Charleroi 
 
The proposed project workflow is schematically depicted in Figure 6. All stages in this work will be 
performed according ISO15189 standards. 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 6. Study design. 
 

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4.1 
Prospective track 
Regarding the prospective track (Figure 6), each month from September 2020 to May 2022 (20 
months), twelve laboratories listed in Table 1 committed to send the first 15 unduplicated isolates of S. 
pneumoniae collected from patients meeting the following criteria:  
Inclusion criteria: 
 
Patients living in Belgium at the time of the study, 
 
from whom unduplicated S. pneumoniae isolates were collected in routine practices,  
 
from non-invasive upper (e.g.: pus/fluid from nasal sinus or from otitis media fluid) or lower 
respiratory tract (e.g. sputum bronchial or endotracheal aspirate, bronchoalveolar fluid (BAL),…) 
clinical samples. 
 
from patients diagnosed with pneumonia, sinusitis and otitis.  
Exclusion criteria: 
 
Patients for whom S. pneumoniae was simultaneously isolated from blood or another usually 
sterile specimen.  
Consequently, a theoretical maximal amount of 3,600 non-invasive S. pneumoniae isolates will be 
prospectively collected, although the actual number will probably be lower given the lower prevalence 
of NIPD during the summer period. For every shipped strain (with associated metadata) during the study 
period, the laboratory will be compensated by payment of €20. 
The transfer of the isolates (under dry ice) from hospital laboratories to the “Sciensano Unit 
‘Antibiotics & Resistance’ (Site Engeland - Engelandstraat 642, 1180 Uccle)” will be organised in the 
last week of every month, using the Sciensano internal sample transport service. Isolates will be 
accompanied by an electronic record sheet, listing the following metadata upon availability :  
 
isolate number 
 
Hospitalised (HOS), residing in a long-term care facility (LCF), ambulatory (AMB), or other 
(to be specified). 
 
admission date (hospitalised patients only)  
 
date of specimen collection 
 
patient age 
 
patient sex 
 
sample source including collection method 
 
clinical diagnosis 
 
vaccination status of the patient for Streptococcus pneumoniae 
 
For bacterial cultures from sputum, the amount of epithelial cells and white blood samples 
 
Other potential respiratory pathogens isolated (e.g. H. influenzae, M. pneumoniae, …) 

10 
 
 
For all isolates sent, following analyses will be performed at Sciensano:  
 
Identification and confirmation of isolates by reference methods prior to use. 
 
Capsular typing using an FT-IR based method that will be developed in the scope of the 
project (section 5.4). 
 
Characterization of NeSP strains by gene sequencing of the cps locus. 
 
Antibiotic susceptibility tests by microdilution (MIC). 
 
Whole-Genome Sequencing (WGS) for multi-drug resistance and non-typeable strains or if 
clonality of the same serotypes originating from different, but geographically linked, patients 
needs to be assessed. 
4.2 
Retrospective track 
As described in Section 1.4, Sciensano holds a collection of >5,000 NIPD strains (1995-2015) for 
which extracted gDNA, complete MIC profile and associated metadata are already available. From every 
uneven year, we will randomly select 150 isolates, without bias towards age and gender. On these 
strains, we will perform FT-IR capsular typing, which will have been developed during the first months 
of this project (section 5.4). Typing of the isolates of the retrospective study will be performed in the 
summer period of 2021, when few isolates of the ongoing prospective study (2020-2022) are expected.  
 
 

11 
 
 
4.3 
Gannt Chart 
 
 
2019 
2020 
2021 
2022 
 
 
 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
PREPARATORY STEPS 
Ethical / GDPR approval  
 
 
 
 
 
 
 
 
 
 
 
 
 
FT-IR Capsular typing 
 
 
 
 
 
 
 
 
 
 
 
 
 
Test development 
 
 
 
 
 
 
 
 
 
 
 
 
 
Test validation 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol communication 
 
 
 
 
 
 
 
 
 
 
 
 
 
Shipment preparations 
 
 
 
 
 
 
 
 
 
 
 
 
 
Database establishment 
 
 
 
 
 
 
 
 
 
 
 
 
 
MIC plate design and order 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PROSP/RETROSP. TRACK 
Prospective Strain collections 
 
 
 
 
 
 
 
 
 
 
 
 
 
Strain conservation 
 
 
 
 
 
 
 
 
 
 
 
 
 
Capsular typing 
 
 
 
 
 
 
 
 
 
 
 
 
 
Typing of NeSP isolates 
 
 
 
 
 
 
 
 
 
 
 
 
 
MIC testing 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Retrospective capsular typing  
 
 
 
 
 
 
 
 
 
 
 
 
 
WGS analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DATA ANALYSIS 
DISSEMINATION 
 
Statistical analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Intermediate report* 
 
 
 
 
 
 
 
 
 
 
 
 
 
Final report 
 
 
 
 
 
 
 
 
 
 
 
 
 
* Intermediate reports will not be published publicly, and can only be consulted by participating hospitals and funding 
partners.  
 
M1. Ethical Approval granted 
M2. FT-IR based capsular typing test validated for 24 pneumococcal serotypes 
M3. Protocol approved by all partners  
M4. Preparatory phase finished, with Sensititre™ plates received, database and lab shipments completely prepared 
M5. Capsular typing completed for selected non-invasive S. pneumoniae isolates (1995-2014)  
M6. Capsular typing and MIC testing completed for all NIPD isolates collected during 20 months (2021-2022) 
M7. Final statistical analyses on complete dataset finished 
Publication 1 (provisional title): An FT-IR based test for fast cps typing in Streptococcus pneumoniae.  
Publication 2 (provisional title): The genetic diversity of Belgian invasive and non-invasive Streptococcus pneumoniae strains 
in an international context.  
Publication 3 (provisional title): Distribution of serotypes and drug resistance profiles of Belgian non-invasive Streptococcus 
pneumoniae strains in light of national vaccination campaigns 
 
 
P

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5 Microbiology 
5.1 
Sampling 
Collecting laboratories will: 
 
prepare stock cultures from overnight pure cultures on blood agar plates.  
 
confirm S. pneumoniae identification by a slide agglutination test with specific antisera on the 
colonies, which will subsequently be transferred to a storage tube. A sufficient number of pre-
filled ready-to-use tubes will be provided. 
 
prepare as dense suspensions as possible (preferably the culture from one blood agar plate) in 
1-ml-aliquots of sterile BHI broth containing glycerol (10% v/v) in suitable screw-cap tubes, and 
vortexed thoroughly.  
 
quick freeze (e.g. in liquid nitrogen) immediately after vortexing (if part of the general lab 
routine), and store tubes preferably at  -70°C. 
5.2 
Storage 
Isolates will be kept at  -70°C. Cultures are stored in Brain Heart Infusion (BHI) broth containing 
glycerol (10% v/v). For retrieval of frozen stock cultures, the inoculum will be subcultured on sheep blood 
agar in 5% CO2. 
5.3 
Confirmation of correct identification 
All isolates will be tested for susceptibility to optochin and bile solubility. In case of doubt or negative 
reaction, a slide agglutination test (e.g. Slidex pneumo-Kit, BioMérieux) as well as their MALDI-TOF 
(Biotyper, Bruker) profile will be examined.  
5.4 
Typing of cps genes 
Capsular typing is performed according to the traditional Quellung reaction. However, in this project 
we will investigate the use of Fourier transform infrared (FT-IR) spectroscopy to predict the serotype of 
the most prevalent serotypes [32,33]. It has been reported that discriminatory biochemical fingerprints, 
observed on FT-IR spectra, consistently correlate with sugar-based coating structures that reflect strain 
variation. This will allow to perform capsular typing on a medium-throughput scale (96-well plate format), 
at less than €5 consumable cost per sample. The test will be validated, and published, in collaboration 
with the NRC for pneumococcal diseases. A downside of this test is that it relies on relative (comparative) 
measurements, with a large number of controls (n=24) being needed in every run. This implies 
collaboration with the NRC to traditionally type all strains that were nontypable using FT-IR. 
To type nonencapsulated strains, the cps locus of the strain will be sequenced to classify the strain 
in group I (mutation or deletion in cps genes) or group II (replacement of the cps genes) NeSP. For this 
last group, 2 subgroups can be distinguished in function of genes that replace the cps locus. In the 
NCC1 subgroup (null capsule clade 1), the pspK gene is present, while in the NCC2 subgroup the cps 
genes are replaced by aliC and aliD. 

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5.5 
Drug susceptibility testing 
The antibiotic susceptibility test will be performed by broth microdilution in cation-adjusted Mueller-
Hinton broth with 2-5% (v/v) lysed horse blood, using custom-made Sensititre™ plates (Thermo 
Scientific, USA) containing lyophilized antibiotics. The final volume of broth in each of the wells of the 
microdilution plates is 50 µl, and the inoculum 5 x 104 CFU. The inoculum will be prepared by direct 
suspension of colonies grown for 18-20 hours in 5% CO2 on sheep blood agar in saline. Inoculum 
adjustment will be done with a photometric device. Incubations will be carried out at 35°C in an ambient 
air incubator, and MICs will be read after 20-24 hours using Vizion™ technology. The following strains 
will be used as first-line control: S. pneumoniae ATCC 49619, and S. pneumoniae TPN.881 (strain F9 
from study 25000/BE-96-01; M phenotype). 
In this surveillance study, we will test the antibiotics listed in the table below. 
 
 
MIC testing 
Antibiotic 
Range 
(µg/ml) 
No. 
Dilutions 
Penicillin G 
0.015-8 
10 
Amoxicillin 
0.12-4 
6 
Moxifloxacin 
0.004-8 
11 
Amoxicillin/Clavulanic acid  2:1 
2-16 
4 
Cefuroxime 
0.5-8 
5 
Cefotaxime 
0.016-4 
9 
Clindamycin 
0.016-4 
9 
Erythromycin 
0.25-4 
5 
Meropenem 
0.016-4 
10 
Levofloxacin 
0.008-4 
10 
Tetracycline 
0.25-8 
6 
Imipenem 
0.5-4 
4 
Trimethoprim/Sulfmethoxazole 
0.06-2 
6 
 
5.6 
Whole-Genome Sequencing of selected isolates 
Whole-Genome Sequencing (WGS) will be performed for multi-drug resistant and non-typeable 
strains or if clonality of the same serotypes originating from different, but geographically linked, patients 
needs to be assessed. 
In this project, we budget the sequencing of 300 strains, the selection of which will be made in 
collaboration with the NRC for pneumococcal diseases to maximize genetic insights in the diversity of 
circulating IPD and NIPD serotypes. 

14 
 
 
For every selected strain, gDNA will be extracted using the automated MagCore™ platform (Atrida, 
NL) using standard procedures for gram-positive bacteria. Illumina MiSeq™ sequencing will be 
performed in-house, according to internally validated procedures. In brief, high-quality sequences are 
mapped onto a single reference genome, S. pneumoniae ATCC700669. We will use the bioinformatic 
pipeline designed by the NRC, to maximize the comparability with IPD isolates.  
5.7 
Data processing and transmission of the results 
For every NIPD isolate, all typing data will be collected along with its metadata in a central database 
using a standardized software format (e.g. Microsoft Access), which will be password protected, with 
aggregated and anonymized data accessible to the participating laboratories.  
 
6 Statistics 
6.1 
Calculation of the sample size 
To estimate the number of samples to be sent by each hospital, we calculated a total sample size 
based on the main objective; estimating the proportions of circulating serotypes. Based on an estimated 
serotype distribution of: PCV7= 7.5%, serotype 19A= 7.5%, serotype 3 =5%, 4 additional serotypes 
included in vaccine PCV13 =30%, serotypes not included in the vaccine non-PCV13 = 50%, we would 
need a total of 2250 samples to be able to obtain these estimates within 95% confidence intervals with 
a width of ± 20 during the whole study period (2020-2022). This translates into approximately 2 samples 
per week from each hospital for the 20 months period (ie total sample size/(number of hospital*weeks 
of study): 2250/(12*80) ≅ 2).  
For the first secondary objective (establishing a statistically significant shift in serotypes from 
serotypes included in the PCV-13 vaccine to serotypes not included in the PCV-13 vaccine between 
retrospective and prospective samples), we estimated the necessary sample size to detect a 20% 
decrease in PCV-13 serotypes (PCV7-serotypes, 19A, 3, 4 additional PCV13) (or 20% increase in non-
PCV-13 serotypes) at n=1500 for each time point (1995-2015 and 2020-2022). 
6.2 
Evaluation 
Our final goal is to investigate Belgian trends in serotype distribution in the non-invasive 
pneumococcal population. The statistical data analysis will be performed with Stat10 software (Stata 
Statistical Software: Release 13. College Station, TX), in collaboration with Sciensano’s Epidemiology 
Service. 
Using data from the Prospective/Retrospective surveillance track: 
 
Chi-square trend analysis of circulating serotypes within the non-invasive S. pneumoniae 
population, integrating retrospective data (1995-2014) with prospectively collected data (2020-
2022). Data will be compared with the serotype dynamics of IPD, annually published by the 
NRC for pneumococcal diseases.  

15 
 
 
 
Measurement of association strength within each stratum of age, gender and infection type by 
the prevalence ratios with exact Fisher tests for statistical significance. Particular attention will 
be given to NeSP isolates, their association with patient type and their influence on drug 
resistance. 
 
Chi-square trend analysis of antimicrobial resistance for all tested drug classes, in relation to 
historic data (1995-2013) and in association with serotype.  
 
Integrating metadata with WGS, we will perform phylogenetic analysis of sequenced isolates in 
their national and international context [13,14], and identification of genetic determinants of 
antibiotic resistance. 
 
7 Access to source data and data in general 
Neither of the funding partners can have a role in data collection and interpretation. However, funding 
partners can request at any time intermediate results or reports. The final report will be put under a six 
months embargo before open access publishing in Summer 2023. 
 
 
8 Monitoring and quality assurance 
All laboratory analyses will be performed according to ISO15089 standards. An internal project board 
consisting Alexandra Vodolazkaia (MD, PhD), Ioannis Passaris (Ir, PhD), Pieter-Jan Ceyssens (Ir., PhD) 
and Raymond Vanhoof (MD, PhD) will monthly follow up experimental results.  
 
9 Adverse events 
Not applicable.  
 
10 Ethics 
Upon approval of the project by at least two contact partners, a general and specific ethical 
application will be filed, in collaboration with Sciensano’s legal department and GDPR officer. Approval 
of local and ethical committees will be sought. 
 
 
 
 
 

16 
 
 
11 Budget 
 
PERSONNEL 
 
Description 
% Effort dedicated to 
this study 
Institutional salary (3 
years) 
TOTAL 
 
Coordinating Scientist 
0.6 FTE 
€ 276,372 
€ 165,823.2 
 
Statistician  
0.2 FTE 
-* 
  € 0 
 
Lab Technician 
0.8 FTE 
€ 135,312 
€ 108,249.6 
 
SUBTOTAL 
€ 274,072.8 
 
STUDY/LAB SUPPLIES   
  
Description 
Unit Cost 
# of Units 
TOTAL 
  
  
  
Strain handling (collection, 
transport) 
€ 21.50 
3,600 
€  77,400 
Strain investigation (ID, MIC 
testing, geno-serotyping) 
€ 24.03 
3,600 
€ 86,508 
Retrospective study 
€9.60 
1,500 
€ 14,400 
WGS of 300 strains 
€ 94.50 
300 
€ 28,350 
  
  
SUBTOTAL 
€  206,658 
 
ADDITIONAL COSTS and FEES 
 
  
Description 
Unit Cost 
# of Units 
TOTAL 
  
FT-IR development 
€ 6,500 
1 
€ 6,500 
  
Publication fees 
€ 4,000 
3 
€ 12,000 
 
Administration 
€ 5,000 
1 
€ 5,000 
  
 
 
SUBTOTAL 
€ 23,500 
DIRECT COSTS - SUBTOTAL 
€ 504,230,8 
OVERHEADS (20%) 
€ 100,846.2 
TOTAL STUDY BUDGET 2020-2022 
(will be equally split among participating companies) 
€ 605,076.9 
MAXIMAL TOTAL BUDGET REQUESTED PER FUNDING 
PARTNER** 
€ 302,538.5 
MAXIMAL REQUESTED ANNUAL REQUESTED BUDGET** 
€ 100,846.2 
To avoid any conflict of interest, this proposal is send at the same time to the three companies producing 
pneumococcal vaccines, being Pfizer, GSK and MSD. This project will only be launched if at least 2 out of 3 
companies are willing to participate and share the associated costs. 
 
 
 

17 
 
 
12 References 
1. 
O'Brien KL, Wolfson LJ, Watt JP, et al. 2009. Burden of disease caused by Streptococcus pneumoniae in children 
younger than 5 years global estimates. Lancet 374:893-902. 
2. 
Welte T, Torres A, Nathwani D. Clinical and economic burden of community-acquired pneumonia among adults in 
Europe. Thorax. 2012;67:71–9.  
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Blasi F, Mantero M, Santus P, Tarsia P. Understanding the burden of pneumococcal disease in adults.Clin Microbiol 
Infect. 2012;18(Suppl 5):7–14.  
4. 
Vaccinatie van kinderen en adolescenten tegen pneumokokken. Advies n° 8813. Hoge Gezondheidsraad. 2015.  
5. 
Olarte L, Kaplan SL, Barson WJ, et al. 2017. Emergence of Multidrug-Resistant Pneumococcal Serotype 35B among 
Children in the United States. J Clin Microbiol. 55(3): 724-734.  
6. 
Janoir C, Lepoutre A, Gutmann L, et al. 2016. Insight Into Resistance Phenotypes of Emergent Non 13-valent 
Pneumococcal Conjugate Vaccine Type Pneumococci Isolated From Invasive Disease After 13-valent Pneumococcal 
Conjugate Vaccine Implementation in France. Open Forum Infect Dis. 3(1):20.  
7. 
Navarro-Torné A, Dias JG, Hruba F, et al., 2015. Risk factors for death from invasive pneumococcal disease, Europe, 
2010. Emerg Infect Dis. 21(3):417-25. 
8. 
Valentino MD, McGuire AM, Rosch JW, et al. 2014. Unencapsulated Streptococcus pneumoniae from conjunctivitis 
encode variant traits and belong to a distinct phylogenetic cluster. Nature Commun 5:5411.  
9. 
Keller LE, Robinson DA, McDaniel LS. 2016. Nonencapsulated Streptococcus pneumoniae: Emergence and 
Pathogenesis. MBio. 7(2):e01792.  
10. Said MA, Johnson HL, Nonyane BAS, Deloria-Knoll M, O'Brien KL, AGEDD Adult Pneumococcal Burden Study Team, 
et al. 2013. Estimating the burden of pneumococcal pneumonia among adults: a systematic review and meta-analysis 
of diagnostic techniques. PLoS ONE  8: e60273. 
11. Broulette J, Yu H, Pyenson B, Iwasaki K, Sato R. The incidence rate and economic burden of community-acquired 
pneumonia in a working-age population. Am Health Drug Benefits. 2013 Sep;6(8):494-503. 
12. Rosón B, Fernández-Sabé N, Carratalà J, Verdaguer R, Dorca J, Manresa F, Gudiol F. 2004. Contribution of a urinary 
antigen assay (Binax NOW) to the early diagnosis of pneumococcal pneumonia. Clin Infect Dis. 38(2):222-6.  
13. Shoji H, Domenech A, Simonetti AF, et al. 2018. The Alere BinaxNOW Pneumococcal Urinary Antigen Test: Diagnostic 
Sensitivity for Adult Pneumococcal Pneumonia and Relationship to Specific Serotypes. J Clin Microbiol. 56(2). pii: 
e00787-17.  
14. Jain S, Self WH, Wunderink RG, et al. 2015. Community-Acquired Pneumonia Requiring Hospitalization among U.S. 
Adults.N Engl J Med. 373(5):415-27. 
15. Blommaert A, Hanquet G, Willem L, Theeten H, Thiry N, Bilcke J, Verhaegen J, Beutels P. Use of pneumococcal 
vaccines in the elderly: an economic evaluation. Health Technology Assessment (HTA) Brussels: Belgian Health Care 
Knowledge Centre (KCE). 2016. KCE Reports 274. D/2016/10.273/79. 
16. Bonten MJM, Huijts SM, Bolkenbaas M, Webber C, Patterson S, Gault S, et al. Polysaccharide Conjugate Vaccine 
against Pneumococcal Pneumonia in Adults. New England Journal of Medicine. 2015;372(12):1114-25. 
17. Benfield T, Skovgaard M, Schønheyder HC, Knudsen JD, Bangsborg J, Østergaard C, et al. Serotype Distribution in 
Non-Bacteremic Pneumococcal Pneumonia: Association with Disease Severity and Implications for Pneumococcal 
Conjugate Vaccines. PloS one. 2013;8(8):e72743 
18. Ochoa-Gondar O, Vila-Corcoles A, Rodriguez-Blanco T, GomezBertomeu F, Figuerola-Massana E, Raga-Luria X, et 
al. Effectiveness of the 23-valent pneumococcal polysaccharide vaccine against community-acquired pneumonia in the 
general population aged >/= 60 years: 3 years of follow-up in the CAPAMIS study. Clinical infectious diseases : an 
official publication of the Infectious Diseases Society of America. 2014 Apr;58(7):909-17. 
19. Horácio AN, Diamantino-Miranda J, Aguiar SI, Ramirez M, MeloCristino J, the Portuguese Group for the Study of 
Streptococcal I. The Majority of Adult Pneumococcal Invasive Infections in Portugal Are Still Potentially Vaccine 
Preventable in Spite of Significant Declines of Serotypes 1 and 5. PloS one. 2013;8(9):e73704.  
20. Horácio AN, Lopes JP, Ramirez M, Melo-Cristino J, for the Portuguese Group for the Study of Streptococcal I. Non-
Invasive Pneumococcal Pneumonia in Portugal—Serotype Distribution and Antimicrobial Resistance. PloS one. 
2014;9(7):e103092 
21. Kim SH, Bae IK, Park D, et al. 2016. Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae 
Isolates Causing Invasive and Noninvasive Pneumococcal Diseases in Korea from 2008 to 2014. Biomed Res Int. 2016: 
6950482. 
22. O’Brien KL, David AB, Chandran A, et al. 2008. Randomized, controlled, trail efficacy of pneumococcal conjugate 
vaccine against otitis media among Navajo and White Mountain Apache infants. Pediatr Infect Dis J 27:71–73.  
23. Buznach N, Dagan R, Greenberg D. 2005. Clinical and bacterial characteristics of acute bacterial conjunctivitis in 
children in the antibiotic resistance era. Pediatr Infect Dis J 24:823–828. 
24. Vanhoof, R. In vitro study to investigate the antimicrobial activity of various antibiotics against non-invasive clinical 
isolates of Streptococcus pneumoniae collected in Belgium during winter 2012-2013. 2013. (http://bacterio.wiv-
isp.be/reporting/reportspdf/SP3013avec%20couverture.pdf) 
25. Liñares J, Ardanuy C, Pallares R, et al. Changes in antimicrobial resistance, serotypes and genotypes in Streptococcus 
pneumoniae over a 30-year period. Clin Microbiol Infect 2010; 16: 402-410. 
26. Niederman MS, Luna CM. Community-acquired pneumonia guidelines: a global perspective. Semin Respir Crit Care 
Med 2012; 33: 298-310.  
27. Adam HJ, Hoban DJ, Gin AS, et al. Association between fluoroquinolone usage and a dramatic rise in ciprofloxacin-
resistant Streptococcus pneumoniae in Canada, 1997-2006. Int J Antimicrob Agents 2009; 34: 82-85.  

18 
 
 
28. WHO. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 
Geneva, Feb 27, 2017. 
29. Sociéte Belge d'Infectiologie et de Microbiologie. Recommandations de traitements anti-infectieux en milieu hospitalier. 
Belgian Antibiotic Policy Coordination Committee. 2017 
30. Vanhoof R, Camps K, Carpentier M, De Craeye S, Frans J, Glupczynski Y, Goffinet P, Gordts B, Govaerts D, Ide L, 
Lefèvre P, Lontie M, Cartuyvels R, Meunier F, Mulongo B, Philippart I, Surmont I, Van Bossuyt E, Van Eldere J, 
Verhaegen J. 10th survey of antimicrobial resistance in noninvasive clinical isolates of Streptococcus pneumoniae 
collected in Belgium during winter 2007-2008.Pathol Biol (Paris). 2010 Apr;58(2):147-51. 
31. Smith KJ, Zimmerman RK, Lin CJ,  et al. 2008. Alternative strategies for adult pneumococcal polysaccharide 
vaccination: a cost-effectiveness analysis.  Vaccine 26(11):1420-1431 
32. Marimón JM, Ercibengoa M, Santacatterina E, et al. 2016. Single-Step Multiplex PCR Assay for Determining 92 
Pneumococcal Serotypes. J Clin Microbiol. 54(8):2197-200.  
33. Selva L, del Amo E, Brotons P, Muñoz-Almagro C. 2012. Rapid and easy identification of capsular serotypes of 
Streptococcus pneumoniae by use of fragment analysis by automated fluorescence-based capillary electrophoresis. J 
Clin Microbiol 50:3451–3457.  
 
 
13 Changes in Protocol to previous version:  
 
Figure 6 : Changed the phrase ‘Genetic Serotyping’ to ‘Capsular Typing’ 
 
Section 4.1 :  
o 
Deleted from exclusion criteria “Patients for whom other bacterial pathogens were detected 
in sample besides S. pneumoniae. This is to limit the inclusion of asymptomatic carriers in the 
study.” 
o 
Added to inclusion criteria: “from patients diagnosed with pneumonia, sinusitis and otitis.”  
o 
Moved to metadata requirements: “For bacterial cultures from sputum and endotracheal 
aspirate samples, the amount of epithelial cells and white blood samples” 
o 
Added: “For every shipped strain (with associated metadata) during the study period, the 
laboratory will be compensated by payment of €20” 
 
Section 4.3. Changed M2 to “FT-IR based capsular typing test validated for 24 pneumococcal 
serotypes” 
 
 
Section 5.3. S. pneumoniae confirmation was changed to match with methods in the NRC, towards “All 
isolates will be tested for susceptibility to optochin and bile solubility. In case of doubt or negative 
reaction, a slide agglutination test (e.g. Slidex pneumo-Kit , BioMérieux) as well as their MALDI-TOF 
(Biotyper, Bruker) profile will be examined.” 
 
 
Section 5.4; Genetic capsular typing is replaced by FT-IR based typing.” Capsular typing is performed 
according to the traditional Quellung reaction. However, it this project we will investigate the use of 
Fourier transform infrared spectroscopy to predict the serotype of the most prevalent serotypes [32,33]. 
It has been reported that discriminatory biochemical fingerprints observed on FT-IR spectra have been 
consistently correlated with sugar-based coating structures that reflect strain variation. This will allow 
to perform capsular typing on a medium-throughput scale (96-well plate format), at less than €5 
consumable cost per sample. The test will be validated, and published, in collaboration with the 
National Reference Center for Pneumococcal diseases (UZ Leuven, Belgium).” 
 
 
Section 5.5. The table with antibiotics to be tested was adapted, to match the drugs tested at the NRC.  
 
 
Section 5.6; WGS data analyses will be done using the pipeline designed by the NRC at UZLeuven