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Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Brief report

Carriage of Streptococcus pneumoniae in asymptomatic,

community-dwelling elderly in the Netherlands
Anna M.M. van Deursen a,b,c , Menno R. van den Bergh a,b ,
Elisabeth A.M. Sanders a, , on behalf of the Carriage Pilot Study Group
a
Department of Pediatric Immunology and Infectious Diseases, Wilhelmina Childrens Hospital, University Medical Center Utrecht, The Netherlands
b
Spaarne Gasthuis Academie, Spaarne Gasthuis, Hoofddorp, The Netherlands
c
Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Colonization of the upper respiratory tract by Streptococcus pneumoniae is considered prerequisite for
Received 6 July 2015 pneumococcal disease. Despite high rates of pneumococcal disease in elderly, pneumococcal carriage
Received in revised form 4 November 2015 rates are usually below 5% when detected by the conventional culture method.
Accepted 6 November 2015
We assessed pneumococcal carriage in 330 asymptomatic community-dwelling elderly aged 65 years
Available online xxx
and older. While pneumococci were cultured from 25 (8%) individuals, 65 (20%) elderly were positive for
the pneumococcus-specic lytA gene when tested by quantitative-PCR, increasing the overall number of
Keywords:
carriers to 75 (22%). Signicantly more oropharyngeal samples were pneumococci-positive (18% versus
Streptococcus pneumoniae
Colonization
10%, p < 0.001) when tested by the molecular method as compared to nasopharyngeal samples.
Elderly Our ndings indicate that pneumococcal carriage in elderly is higher than previously reported with up
Nasopharynx to 1 in 5 asymptomatic community-dwelling elderly positive for pneumococcal carriage, when detected
PCR by qPCR. The detection of pneumococci by conventional culture alone, greatly underestimates S. pneu-
moniae colonization in elderly.
2015 Elsevier Ltd. All rights reserved.

1. Introduction signicantly increase pneumococcal carriage detection [8]. We

The incidence of invasive pneumococcal disease (IPD) and com-
munity acquired pneumonia (CAP) typically follows a U-shaped
curve, peaking at the extremes of life [1]. In the elderly, Streptococ-
cus pneumoniae is a major cause of CAP with up to 40% mortality
in bacteremic cases [1]. Nasopharyngeal carriage of S. pneumoniae
is seen as prerequisite for pneumococcal disease. The high disease-
rates in children under ve years of age coincide with high rates of
pneumococcal carriage [2], owing to their immature immune sys-
tem. However, despite high incidence of IPD and CAP in elderly,
carriage of S. pneumoniae in aged community dwelling adults is
reported to be below 5% [35] when detected by the WHO recom-
mended conventional culture method of naso- and oropharyngeal
swabs [6]. More advanced molecular techniques advocated to
detect low-density S. pneumoniae colonization [7] have rarely been
applied in the elderly. The standard practice for detecting pneumo-
coccal carriage in children is culture of a nasopharyngeal sample.
In adults, the addition of oropharyngeal samples is known to
recently reported over 30% rates of pneumococcal carriage in
elderly suffering from inuenza-like illness (ILI) and after recovery
combining both culture and molecular methods on samples from
swabs and saliva [9].
Accurate measures of pneumococcal prevalence in the upper
respiratory tract of healthy, community-dwelling elderly is impor-
tant, not only for better understanding on the pathogenesis of
pneumococcal diseases like CAP or IPD, but also to assess the direct
and indirect effect of pneumococcal vaccinations in this population.
Prior to the start of a randomized controlled trial assessing
the efcacy of the 13-valent pneumococcal conjugate vaccine
(Prevenar-13, Pzer, Inc.) against CAP in elderly (the Commu-
nity Acquired Pneumonia immunization Trial in Adults) [10],
we assessed S. pneumoniae colonization in 330 asymptomatic,
community-dwelling elderly aged 65 years and older by con-
ventional culture and molecular methods on both oro- and
nasopharyngeal samples.

2. Methods
Clinical trial registry: VR-6115A1-3014.
Corresponding author. We performed a cross-sectional study in a random sample of 330
E-mail address: l.sanders@umcutrecht.nl (E.A.M. Sanders). asymptomatic elderly aged 65 years and older in the Netherlands.

http://dx.doi.org/10.1016/j.vaccine.2015.11.014
0264-410X/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: van Deursen AMM, et al. Carriage of Streptococcus pneumoniae in asymptomatic, community-dwelling
elderly in the Netherlands. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.11.014
G Model
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Participants were visited at home between December 10, 2007 and
January 30, 2008. At this time, the 7-valent pneumococcal vaccine
had been in use for 1.5 years, for infants born after March 31, 2006
(without a catch-up campaign) [11]. Until 2008, no herd protec-
tion for IPD in elderly had been observed [12]. In the Netherlands,
23-valent pneumococcal polysaccharide vaccine (PPSV23) is only
recommended for high-risk groups (e.g. asplenic people, Hodgkins
lymphoma patients) and the uptake in elderly was negligible (<1%)
[13]. Antibiotic use in the preceding month was an exclusion crite-
rion for the study.
The upper respiratory tract was sampled using two separate
naso- and two separate oropharyngeal swabs. Samples were col-
lected by trained study personnel, immediately stored in Amies
transport medium (Copan, Brescia, Italy) and transferred within
8 hours at room temperature to the Regional Laboratory (Haar-
lem, the Netherlands). Upon arrival at the laboratory, the rst of
each swab type was tested for the presence of S. pneumoniae by
conventional culture methods, as previously described [2]. Pneu-
mococcal serotyping was performed using the capsular swelling
method (quellung reaction) with type-specic antisera (Statens
Serum Institut, Copenhagen, Denmark).
The second of the naso- and oropharyngeal swabs were stored
in transport medium at 70 C until transferred to Pzers labora-
tories for further analyses. After thawing, DNA was extracted from
100 l of each specimen using an ABI 6700 Nucleic Acid Work-
ingstation (Applied Biosystems, CA) and eluted in a nal volume of
100 l. S. pneumoniae was detected by qPCR targeting conserved
regions of the autolysin (lytA) gene [14]. Ten microliters of puried
DNA was added to 10 L 2X Fast PCR mix (Applied Biosystems)
containing primers and probe and DNA fragments were amplied
using the 7900 Fast Real-Time PCR System (Applied Biosystems,
CA). Samples were considered positive for S. pneumoniae when the
lytA-specic signal detected was <45 CT [15]. Samples with high
levels of lytA (CT < 35) were tested in 13 real-time PCR assays to
determine the 13-valent vaccine serotypes (VT). The PCR assays
for serotypes 7F, 9V, and 18C are serogroup specic, not serotype
specic.
3. Results
The median age of the 330 participants was 72.7 years
(interquartile range: 68.779.0) and 156 (47%) were male. Four
subjects (1%) had previously received PPSV23.
Of all subjects, 25 (8%, 95% condence interval (CI): 511%) were
positive for S. pneumoniae by conventional culture (Table 1). If only
naso- or oropharyngeal samples had been collected, only 16 (5%)
individuals would have been identied by conventional culture as
carriers, by either sample type.
Based on qPCR alone, pneumococci were detected in 65 naso-
and oropharyngeal samples (22%, 95% CI: 1827%). By qPCR-
detection signicantly more oro- (n = 58; 18%) than nasopharyngeal
Table 1
Conventional culture and qPCR detection of S. pneumoniae in naso- and oropharyngeal samples from 330 asymptomatic community-dwelling elderly 65 years of age, in the
Netherlands.
Conventional culture
n (%)
Nasopharyngeald 16 (5)
Oropharyngeal 16 (5)
Both naso- and oropharyngeal 7 (2)
Either naso- or oropharyngeal 25 (8)
NA, not applicable.
a
Results for lytA qPCR; any signal for lytA (CT value <45) was considered positive for the presence of S. pneumoniae (6, 8, 9).
b
Proportion of samples with corresponding results in both tests over the total number of samples tested.
c
McNemar test for paired samples.
d
Results from 6 nasopharyngeal specimens were unavailable; concordance was calculated for 324 samples.
Please cite this article in press as: van Deursen AMM, et al. Carriage of Streptococcus pneumoniae in asymptomatic, community-dwelling
elderly in the Netherlands. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.11.014
(n = 32; 10%) samples were found to be positive for lytA (P < 0.001).
The mean CT value was 36.05 (range: 2440) and 36.31 (range:
2544) for naso- and oropharyngeal samples, respectively. Com-
bining both conventional culture and qPCR 75 individuals (23%,
95% CI: 1928%) were dened as positive for S. pneumoniae in
either their naso- or oropharyngeal sample or both. Carriage of
S. pneumoniae by either conventional culture or qPCR was lower
in the eldest age group (29.2% in 6574 years and 13.6% in 75
years of age). Carriage rates between subjects with and those with-
out self-reported comorbidities were comparable; 22.1% and 23.4%
respectively.
In 34 of the 75 subjects positive for S. pneumoniae (45.3%),
serotyping was performed by either quellung reaction (16 naso-
and 16 oropharyngeal sample isolates from 25 subjects) or PCR (11
naso- and 16 oropharyngeal samples with CT of lytA <35 from 21
subjects). Over fty percent (n = 18, 51.4%) of these subjects carried
a PCV13-VT serotype, 37.1% (n = 13) a non-VT serotype and 11.4%
(n = 4) were nontypable. In 2 of the 34 subjects (5.9%) more than
one VT was detected in the oropharyngeal samples (serotypes 1 and
19A; and serotypes 1, 19F, and serogroup 7). Of the VT serotypes,
serotype 3 (n = 4; 11.8%) and 19F (n = 3; 8.8%) were most preva-
lent. Of the cultured non-VT, serotype 35F (n = 5; 14.3%) was most
abundant.
4. Discussion
Our results conrm the high additional value of molecular tech-
niques over conventional culture for detecting the presence of
S. pneumoniae in the upper respiratory tract in general. This is
particularly true for older adults with low density pneumococcal
carriage that may be missed by culture alone and in the polymi-
crobial oropharyngeal specimens in particular, when tested with
a sensitive qPCR assay targeting the lytA gene (18% qPCR-positive
vs 5% culture-positive; P < 0.001). We and others have previously
reported on the high specicity of this method, thus we are con-
dent that our results reect a genuine presence of pneumococcal
DNA in the specimens tested [6,8,9,14,15].
Our results show a point prevalence pneumococcal carriage rate
of up to 23% in asymptomatic community-dwelling elderly based
on both conventional culture and qPCR without previous culture
enrichment, which is higher than previous reports using conven-
tional culture alone. Krone et al. reported a pneumococcal carriage
rate of 14% in older adults with ILI based on qPCR-detection of
lytA in culture-enriched naso- and oropharyngeal samples, using
a lower cut-off for sample positivity of CT [9]. When applying the
same criteria to our data, we still nd a slightly higher carriage rate
of 18% (n = 59) although the difference was not signicant between
studies (Fisher exact test, p = 0.222).
In the present study, 7 of 32 (22%) culture-positive samples were
negative when tested by qPCR. Conversely, there were also ve
samples (one naso- and four oropharyngeal samples) with rela-
tively strong lytA CT values (CT < 35), with corresponding cultured
qPCRa Concordanceb P valuec
n (%)
32 (10) 92% P = 0.001
58 (18) 85% P < 0.001
19 (6) NA NA
71 (22) NA NA
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samples for S. pneumoniae. This discordance might be explained by
the fact that our culture and qPCR results were derived from sepa-
rate swabs and a low carriage density in older adults. The relatively
weak CT values observed by us seem to conrm overall low-density
colonization, in line with reported low-density nasopharyngeal S.
pneumoniae colonization in asymptomatic adults Moreover, only
a small volume of transport medium from the swabs was eval-
uated with qPCR. The discordant results between culture and
qPCR therefore most likely represent the random presence or
absence of pneumococci in these low density samples from sepa-
rate swabs. Evaluation of saliva enhances pneumococcal carriage
rates. We previously showed that in saliva samples from older
adults, culture-enrichment doubled detection of S. pneumoniae
when tested for pneumococcal lytA and Pia [9]. In the present
study, we did not culture enriche naso- and oropharyngeal samples
before molecular evaluation and did not evaluate saliva. The low
sensitivity of bacterial culture in elderly also shows the problem
in detecting S. pneumoniae in polymicrobial samples, in particu-
lar in case of low abundance. Oropharyngeal samples are difcult
to process due to the presence of many competing species, mak-
ing pneumococcal isolation by conventional culture a laborious
and difcult process [16]. The higher number of carriers being
more commonly detected from oropharyngeal samples as com-
pared to nasopharyngeal samples [8,9] strongly suggests that the
pneumococcal carriage niche may be shifting with age, from the
upper nasopharynx to the oropharynx. Overall, results of this
study suggest that conventional culture alone strongly underesti-
mates rates of pneumococcal carriage in the elderly and underlines
an urgent need for sensitive and specic, culture-independent
methods for surveillances on pneumococcal carriage in aged
adults.
5. Conclusion
Our results indicate that pneumococcal carriage rates in elderly
are signicantly higher than contemporary reported. Conventional
culture methods greatly underestimate pneumococcal carriage
rates in the elderly indicating an urgent need for sensitive and
specic culture-independent methods for surveillance on S. pneu-
moniae in aged adults.
Funding
This study was funded by a research grant from Pzer Pharma-
ceuticals.
Acknowledgements
We thank all the study participants for their time and commit-
ment. We acknowledge the study team at the Linnaeus Institute,
Spaarne Hospital, Hoofddorp, the Netherlands for their dedica-
tion and work. We would also like to thank the laboratory staff
of Regional Laboratory of Public Health, Haarlem and Pzer labo-
ratories, US, for their dedication and work. Lastly, we would like
to thank A. Wyllie for her contribution in writing this manuscript.
This manuscript is dedicated to the memory of Dr. R. Veenhoven,
investigator at Spaarne Gasthuis in Hoofddorp, the Netherlands,
who contributed substantially to the planning of the design and
the execution of this study.
Please cite this article in press as: van Deursen AMM, et al. Carriage of Streptococcus pneumoniae in asymptomatic, community-dwelling
elderly in the Netherlands. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.11.014
3
Conicts of interest: ES reports receipt of research funding from
Pzer and GlaxoSmithKline plc, and participation in independent
data monitoring committees for Pzer and GlaxoSmithKline plc (all
fees paid to the institution). ADs research funding is partially sup-
ported by grants provided to Spaarne Gasthuis by Pzer. MB was
involved in coordinating and executing studies sponsored by Glax-
oSmithKline Biologicals and Pzer, Inc. and received payment for a
lecture by GlaxoSmithKline Biologicals.
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