Death or survival from invasive pneumococcal
disease in Scotland: associations with serogroups
and multilocus sequence types
Donald Inverarity,13 Karen Lamb,24 Mathew Diggle,31 Chris Robertson,2,8
David Greenhalgh,2Tim J. Mitchell,1Andrew Smith,4
Johanna M. C. Jefferies,5,6,7Stuart C. Clarke,5,6,7Jim McMenamin8
and Giles F. S. Edwards3
Received 8 December 2010
Accepted 3 March 2011
1Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences,
University of Glasgow, 120 University Place, Glasgow G12 8QQ, UK
2Department of Mathematics and Statistics, University of Strathclyde, Glasgow G1 1XH, UK
3Scottish Haemophilus, Legionella, Meningococcal and Pneumococcal Reference Laboratory
(SHLMPRL), Stobhill General Hospital, Glasgow G21 3UW, UK
4College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
5Division of Infection, Inflammation and Immunity, University of Southampton School of Medicine,
Southampton SO16 6YD, UK
6Health Protection Agency, Southampton SO16 6YD, UK
7Southampton NIHR Respiratory Biomedical Research Unit, Southampton SO16 6YD, UK
8Health Protection Scotland, Clifton House, Clifton Place, Glasgow G3 7LN, UK
We describe associations between death from invasive pneumococcal disease (IPD) and particular
serogroups and sequence types (STs) determined by multilocus sequence typing (MLST) using
data fromScotland. All IPD episodes where blood or cerebrospinal fluid (CSF) culture isolates were
referred to the Scottish Haemophilus, Legionella, Meningococcal and Pneumococcal Reference
Laboratory (SHLMPRL) from January 1992 to February 2007 were matched to death certification
records by the General Register Office for Scotland. This represented 5959 patients. The median
number of IPD cases in Scotland each year was 292. Deaths, from any cause, within 30 days of
pneumococcal culture from blood or CSF were considered to have IPD as a contributing factor.
Eighthundred and thirty-threepatientsdiedwithin30 daysofculture ofStreptococcus pneumoniae
from blood or CSF [13.95%; 95% confidence interval (13.10, 14.80)]. The highest death rates
were in patients over the age of 75. Serotyping data exist for all years but MLST data were only
available from 2001 onward. The risk ratio of dying from infection due to particular serogroups or
STs compared to dying from IPD due to all other serogroups or STs was calculated. Fisher’s exact
test with Bonferroni adjustment for multiple testing was used. Age adjustment was accomplished
using the Cochran–Mantel–Haenszel test and 95% confidence intervals were reported.
Serogroups 3, 11 and 16 have increased probability of causing fatal IPD in Scotland while
serogroup 1 IPD has a reduced probability of causing death. None of the 20 most common STs
were significantly associated with death within 30 days of pneumococcal culture, after age
adjustment. We conclude that there is a stronger association between a fatal outcome and
pneumococcal capsular serogroup than there is between a fatal outcome and ST.
Abbreviations: CI, confidence interval; CSF, cerebrospinal fluid; IPD, invasive pneumococcal disease; MLST, multilocus sequence typing; RR, risk ratio;
SHLMPRL, Scottish Haemophilus Legionella Meningococcal and Pneumococcal Reference Laboratory; ST, sequence type.
3Present address: Department of Microbiology, Monklands Hospital, Monkscourt Avenue, Airdrie ML6 0JS, UK.
4Present address: MRC Social and Public Health Sciences Unit, 4 Lilybank Gardens, Glasgow G12 8RZ, UK.
1Present address: Queen’s Medical Centre, Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Derby Road, Nottingham
NG7 2UH, UK.
Journal of Medical Microbiology (2011), 60, 793–802
028803G2011 SGMPrinted in Great Britain793
Infections due to Streptococcus pneumoniae (the pneumo-
coccus) remain a substantial source of morbidity and
mortality in both developing and developed countries
despite a century of research and the development of
therapeutic interventions such as multiple classes of anti-
biotics and vaccination. The World Health Organization
estimates that in developing countries 814000 children
under the age of five die annually from invasive pneu-
mococcal disease (IPD) (Scott, 2007), with an estimated 1.6
million deaths affecting all ages globally (WHO, 2007).
Case fatality rates for pneumococcal meningitis cases are
generally higher than non-meningitis cases (Ru ¨ckinger
et al., 2009), and bacteraemic pneumococcal meningitis has
a poorer outcome than non-bacteraemic pneumococcal
meningitis (Carrol et al., 2008). The mortality rate at
14 days from pneumococcal bacteraemia is generally
around 17% (Yu et al., 2003) although at 30 days it has
been found to be as high as 33% in urban Scotland
(Maddox & Winter, 2003). In patients with IPD, age over
65, underlying chronic disease, immunosuppression and
disease severity have been significantly associated with
increased mortality in multivariate analyses (Alanee et al.,
2007; Yu et al., 2003).
Several recent studies have identified associations between
pneumococcal serotypes and patient outcomes from IPD.
Serotype 3 has been shown to be associated with an
increased relative risk of death and serotype 1 a lower
relative risk of death in Denmark (Harboe et al., 2009). A
recent meta-analysis identified serotypes 1, 7F and 8 as
being associated with decreased relative risk of death due to
pneumococcal pneumonia while serotypes 3, 6A, 6B, 9N
and 19F were associated with increased relative risk of
death (Weinberger et al., 2010).
The primary aim of this study was to identify an associa-
tion between any of the serogroups identified as causing
IPD in Scotland and death at 30 days after culture of
pneumococci. The secondary aim was to determine if any
sequence types are significantly associated with mortality.
In addition, it was of interest to assess associations between
serogroups and sequence types and mortality for different
The IPD episodes referred to in this study relate to clinical isolates
(grown from blood or cerebrospinal fluid, CSF) of S. pneumoniae sent
to the Scottish Haemophilus, Legionella, Meningococcal and
Pneumococcal Reference Laboratory (SHLMPRL) from January
1992 to February 2007, identified at diagnostic microbiology
laboratories in Scotland. At SHLMPRL, these isolates were grown
on Columbia blood agar (Oxoid) at 37 uC under anaerobic
conditions by use of an anaerobic pack (Oxoid) and after a single
subculture were stored at 280 uC on Protect beads (M-Tech
Diagnostics). Optochin susceptibility was confirmed by disc diffusion,
and susceptibilities to penicillin, erythromycin and cefotaxime were
determined using E-test strips (AB Biodisk). Breakpoints published
by the British Society of Antimicrobial Chemotherapy were used
to assess antimicrobial susceptibility as previously described (Cooke
et al., 2010). Isolates were serotyped by an established coagglutination
method (Smart, 1986).
Multilocus sequence typing (MLST) was performed as described
previously (Clarke & Diggle, 2002; Enright & Spratt, 1998; Jefferies
et al., 2003). Briefly, fragments from seven housekeeping genes, aroE,
gdh, gki, recP, spi, xpt and ddl, were amplified from the pneumococcal
lysate with the primers described by Enright & Spratt (1998) by using
a single PCR. The amplified DNA was cleaned as previously described
(Clarke & Diggle, 2002; Sullivan et al., 2006). The cleaned amplified
DNA was then sequenced with the same primer set using the
DYEnamic ET Terminator sequencing kit (Amersham Biosciences).
These procedures were carried out on a liquid handling robotic
platform (MWG-Biotech) and a MegaBACE 1000 DNA sequencer
(Amersham Biosciences). Analysis of the sequence data and sub-
sequent assignment of a sequence type (ST) was performed as
described previously (Diggle & Clarke, 2002).
The database of these isolates, which are stored at SHLMPRL, was
matched to death certification records (Kendrick & Clarke, 1993) by
the General Register Office for Scotland. Only the first isolate received
was used to indicate an episode of IPD in cases when multiple isolates
were received from the same patient. Ethical approval for data
matching was received from North Glasgow University Hospitals
Glasgow Royal Infirmary Research Ethics Committee (REC reference
07/S0704/27). Deaths, from any cause, within 30 days of pneumo-
coccal culture from blood or CSF were considered to have IPD as a
To determine associations between mortality and serogroup or ST,
the risk ratio of dying within 30 days from IPD due to particular
serogroups or STs compared to dying within 30 days from IPD
attributable to all other serogroups or STs was calculated. The risk
ratio (RR) is calculated as (a/(a+b))/(c/(c+d)), where a is the
number of deaths within 30 days of developing IPD attributable to
the serogroup or ST under scrutiny (serogroup i or ST i), b is the
number who survived more than 30 days of serogroup i or ST i IPD, c
is the number of deaths within 30 days attributable to all other
serogroups or STs (i.e. all non-serogroup i or ST i IPD deaths), and d
is the number of survivors of more than 30 days of disease from all
other serogroups or STs. Each serogroup or ST found in IPD was
compared to IPD cases from all other serogroups or STs so that an
estimate of the risk ratio could be obtained for each of the serogroups
or STs relative to all others, rather than compared to a single baseline
serogroup or ST. This approach of floating absolute risk (Easton et al.,
1991) was employed in a study establishing the invasive disease
potential of serotypes and STs among children in Oxford, England
(Brueggemann et al., 2003). A risk ratio of 1 indicates that an
individual with serogroup i or ST i is as likely to die within 30 days of
IPD as an individual with IPD from a serogroup or ST other than i. A
risk ratio of greater than 1 can be interpreted as being indicative of an
increased probability of death within 30 days of IPD due to serogroup
i or ST i invasive disease, whilst a risk ratio of less than 1 is indicative
of a reduced probability for the serogroup or ST to cause death within
To investigate if certain serogroups or STs are associated with a
greater or reduced risk of death within 30 days of IPD, Fisher’s exact
test was used. This test is more appropriate than the x2test of
association as many serogroups and STs are rarely observed in IPD
and some more common serogroups may be rarely observed in IPD
cases with fatal outcomes. The Cochran–Mantel–Haenszel test was
used to carry out adjustment for age when testing the association
between serogroups or STs and mortality. The null hypothesis of this
test is no association between the two categorical variables across all
strata; the alternative hypothesis is that there is an association
D. Inverarity and others
794 Journal of Medical Microbiology 60
between the two variables in at least one of the strata. The Bonferroni
correction factor was used to adjust for multiple testing. Logistic
regression was used to determine whether or not the proportion of
fatalities remained constant over time.
Analyses were performed using R version 2.8.0 (R Development Core
During the study period 833 of the 5959 patients with IPD
[13.95%; 95% CI (13.10, 14.80)] died within 30 days of the
sample submission to SHLMPRL. There were 7 (0.12%)
patients that could not be matched to the data on death
certification records from the General Register Office for
Examination of the number of cases of IPD for each of the
years between 1992 and 2007 showed an increase from a
minimum of 29 cases of IPD observed in 1992 to a
maximum of 697 cases in 2006. The number of IPD cases
appeared relatively low initially and generally increased each
year, until 2000. The apparent increased number of cases of
IPD may be due to improved surveillance rather than a real
increase in the number of cases of IPD in Scotland during
that period. On average, there were approximately 372 cases
(median 292 cases) of IPD in Scotland each year.
The proportion of fatalities attributable to IPD did not
increase over the period of study even though the number
of identified cases increased (Fig. 1). Case fatality decreased
from 24% (7 of 29 cases) in 1992 to 12% (86 of 697 cases)
in 2006. The improved surveillance of IPD in Scotland may
have contributed to this as over time it is likely that isolates
have been referred from more cases associated with less
severe disease than would have been the case in the 1990s,
when isolates from severe or complicated IPD were mainly
referred. Other influences such as improved case manage-
ment cannot be excluded, however.
Fig. 2 shows the distribution of age for the patients with
IPD, with age ranging from 0 to 99 years. IPD appears most
common for those under 10 years of age and those between
70 and 80 years of age. Information regarding age was
missing from 301 patients. Age was categorized into six
strata: 0–4 years, 5–34 years, 35–49 years, 50–64 years, 65–
74 years, and 75 years and over. Of the 5959 patients, 3015
(50.60%) were male and 2913 (48.88%) were female; 31
patients had missing gender information. Fig. 3 shows the
Fig. 1. Plot of the proportion of IPD fatalities each year in Scotland
from 1992 to 2007 (95% confidence intervals shown). A logistic
regression analysis showed that the proportions of fatalities were
not constant over time (P,0.0001) and that the risk of dying within
30 days reduced by 3.92% each year [95% CI (2.48%, 5.99%)].
After adjustment for age group and gender, the results showed
that the risk of dying within 30 days reduced on average by 5.08%
each year [95% (CI 3.19%, 6.94%)].
Fig. 2. Histogram of the age distribution of individuals with IPD in
Scotland from 1992 to 2007.
Age group (years)
50_6465_74 75 & over
Fig. 3. Plot of the proportion of fatalities from IPD each year in
Scotland within gender by age group from 1992 to 2007 (95%
confidence intervals shown).
Risk of death from pneumococcal serogroups and STs
Table 1. Results from Fisher’s exact test and age-stratified Cochran–Mantel–Haenszel test of association between mortality and the
20 most common serogroups
A P-value of less than 0.0025 is required for significance.
SerogroupDied Total Risk ratio Bonferroni-adjusted
P-value Bonferroni-adjusted 95% CI
(stratified by age group)
by age group)
Table 2. Results from Fisher’s exact test and age-stratified Cochran–Mantel–Haenszel test of association between mortality and the
20 most common STs
A P-value of less than 0.0025 is required for significance.
STDiedTotalRisk ratio Bonferroni-adjusted
P-valueBonferroni-adjusted 95% CI
(stratified by age group)
by age group)
D. Inverarity and others
796Journal of Medical Microbiology 60
proportion of fatalities of IPD within gender by age group.
This figure shows that a marginally higher proportion of
females who acquired IPD had fatal outcomes than the
proportion of males who acquired IPD within the age
groups 0–4 years and 35–49 years. For all other age groups,
the opposite is true. Fig. 3 also shows that the highest
proportion of fatalities occurs in the 75 years and over age
group for both genders and the lowest proportion of
fatalities is observed in the 0–4 year old age group.
Thirty-five different serogroups were observed causing IPD
in Scotland between January 1992 and December 2007.
Serogroup 3 had the highest proportion of deaths from
IPD within 30 days with 24% (85 of 349 cases, RR 1.99,
P,0.001) resulting in death. Serogroups 19 and 23 had the
next-highest fatality rates, at 18% (83 of 453 cases, RR
1.39, P50.01) and 15% (59 of 385 cases, RR 1.12, P50.40)
respectively. Serogroup 1 had the lowest rate of fatality
within 30 days, with only 5% (26 of 513 cases, RR 0.33,
P,0.001) of cases resulting in death. Serogroup 7 also has a
relatively low percentage of IPD fatality, at only 8% (23 of
272 cases, RR 0.57, P50.01).
Table 1 shows the risk ratio and Bonferroni-adjusted 95%
confidence interval for the risk of death from each of the 20
most common serogroups identified as causing IPD in
Scotland for both Fisher’s exact test and the age-stratified
There is significant evidence of an association between
mortality and serogroups 1, 3 and 16. Significant evidence of
an association between serogroup 11 and mortality was
found using Fisher’s exact test but not the Cochran–Mantel–
Haenszel test and serogroup 19 was found to be associated
with mortality using the Cochran–Mantel–Haenszel test but
not Fisher’s exact test. Serogroup 3 has a risk ratio of dying
of 1.99 relative to all other serogroups; serogroup 11 has a
risk ratio of 2.11, serogroup 16 has the largest risk ratio of
3.82 and serogroup 19 has a risk ratio of 1.39. Therefore,
thereisanincreased riskofafataloutcomewithin30 daysof
a diagnosis of IPD attributable to any of those serogroups
rather than any other serogroup IPD. Serogroup 1 has a risk
ratio of 0.33. Therefore, there is a reduced risk of fatality
within 30 days of a diagnosis of IPD attributable to this
serogroup compared to all other serogroups.
Serotype information was available from 2003 in Scotland.
IPD from serogroups 6, 7 and 19 was assessed from 2003 to
2007 to investigate associations between the serotypes from
within these serogroups and fatality within 30 days of a
diagnosis of IPD. No significant associations were found
between any of the serotypes and mortality.
There were 371 different STs identified in casesof IPD between
2001 and 2007. Of the 20 most common disease-causing STs,
ST180 has the worst outcome, with 22% of cases (38 of
174 cases, RR 1.98, P,0.001) ending in death at 30 days
post-diagnosis. ST306 has the best outcomes, with only
approximately 3% of cases (8 of 250 cases, RR 0.23,
P,0.001) ending in death.
Table 2 displays the risk ratio and Bonferroni-adjusted 95%
confidence interval for the risk of death of each of the 20
most common STs identified in IPD in Scotland. There is
significant evidence of an association between fatal out-
comes and ST306, ST180 and ST191 from Fisher’s Exact
Test but not from the Cochran–Mantel–Haenszel test. For
ST306 and ST191 there is a reduced risk of death. For ST180
there is an increased risk of death. No significant associa-
tions were found using the Cochran–Mantel–Haenszel test
to adjust for the different age strata. Tables 3 and 4 show
Table 3. STs associated with the serogroups significantly associated with 30 day mortality
SerogroupST (number of isolates)
1306 (243), 227 (96), 1310 (3), 1809 (3), 9 (2), 1882 (2), 2126 (2), 53 (1), 138 (1), 162 (1), 176 (1), 179 (1), 180 (1), 199 (1), 205 (1),
228 (1), 246 (1), 304 (1), 312 (1), 618 (1), 1239 (1), 1247 (1), 1311 (1), 1346 (1), 1597 (1), 2126 (1), 2135 (1), 3229 (1), 3230 (1)
180 (172), 260 (4), 232 (4), 1003 (3), 1468 (3), 1220 (3), 233 (3), 53 (2), missing (2), 1253 (2), 2263 (2), 458 (2), 162 (2), 312 (1),
378 (1), 862 (1), 1300 (1), 1344 (1), 1377 (1), 1682 (1), 1765 (1), 1867 (1), 1887 (1), 2119 (1), 2979 (1)
62 (53), 408 (6), 513 (3), 199 (1), 446 (1), 1180 (1), 1219 (1), 1304 (1)
570 (6), 30 (3), 414 (3), 863 (1), 1382 (1), 2127 (1), 2130 (1), 2262 (1), 2827 (1)
199 (88), 162 (59), 426 (14), 309 (7), 416 (6), 177 (5), 179 (4), 688 (4), 1201 (4), 9 (3), 43 (3), 246 (3), 420 (3), 422 (3), 667 (3),
1718 (3), 191 (2), 193 (2), 236 (2), 276 (2), 419 (2), 423 (2), 424 (2), 645 (2), 654 (2), 686 (2), 1002 (2), 1218 (2), 1359 (2), 53
(1), 58 (1), 66 (1), 81 (1), 124 (1), 156 (1), 165 (1), 176 (1), 251 (1), 271 (1), 306 (1), 312 (1), 395 (1), 425 (1), 438 (1), 450 (1),
459 (1), 462 (1), 476 (1), 482 (1), 494 (1), 644 (1), 655 (1), 697 (1), 799 (1), 826 (1), 839 (1), 994 (1), 1035 (1), 1233 (1), 1258
(1), 1298 (1), 1545 (1), 1757 (1), 2067 (1), 2076 (1), 2220 (1), 2265 (1), 2365 (1), 2370 (1), 3211 (1), 3217 (1)
Table 4. Serogroups associated with the STs significantly
associated with 30 day mortality
STSerogroup (number of isolates)
1 (243), missing (2), 4 (1), 6 (1), 14 (1), 18 (1), 19 (1)
3 (172), missing (1), 6 (1), 33 (1)
7 (164), 6 (2), 19 (2), 4 (1), 5 (1), 14 (1)
1 (96), 6 (1), missing (1)
Risk of death from pneumococcal serogroups and STs
that the STs that are significantly associated with fatal
outcomes are predominantly associated with just one
serogroup. Apart from serogroups 1 and 19 (where
there are several STs represented in each of these
serogroups) there is often a dominant ST for the significant
Considering the serogroups significantly associated with
30 day mortality, within the cases of serogroup 1 there is
no evidence that death from IPD is associated with ST
(P50.12), and compared to ST306, ST227 has a risk ratio
of 1.64 [(95% (CI 0.58, 4.63)], as illustrated in Table 5.
Similarly, within the cases of serogroup 19 there is no
evidence that fatal outcome is associated with ST (P50.97).
Compared to ST199, ST162 has a risk ratio of 1.18 [95% CI
(0.58, 2.40)] and ST426 has a risk ratio of 0.99 [95% CI
Furthermore, no evidence was found of an association
between death from IPD and ST for either serogroup 3
(P50.23) or serogroup 11 (P50.43) comparing IPD from
the dominant ST (ST180 and ST570, respectively) to all
other STs linked to each of these serogroups. There were
too few cases of serogroup 16 IPD to consider associations
between ST and IPD for this serogroup.
Early observations in the history of pneumococcal research
suggested that particular serotypes had a propensity for
more severe disease manifestations and that some serotypes
were more commonly associated with a fatal outcome. In
the pre-antibiotic era, serotype 3 pneumococcal pneu-
monia was associated with high case fatality rates and
serotype 1 with lower case fatality rates when no treatment
other than symptomatic relief was administered (Avery
et al., 1917; Cowan et al., 1932). The introduction of
penicillin had less effect on case fatality rates from serotype
3-associated pneumococcal pneumonia than case fatality
rates due to other serotypes (Austrian & Gold, 1964;
Macfarlane et al., 1982).
In Sweden, greater disease severity has been associated with
serotypes 3, 6A, 6B, 19A and 19F while in the same study,
serotypes 1, 4 and 7F had the least severe disease (Sjo ¨stro ¨m
et al., 2006). Serotypes 3, 6A and 19F also had high case
fatality rates in this study (Sjo ¨stro ¨m et al., 2006) while
serotype 19A alone had a high case fatality rate in another
Swedish study (Berg et al., 2006). Serotypes 1 and 7F had
low case fatality rates in both Swedish studies (Berg et al.,
2006; Sjo ¨stro ¨m et al., 2006). Recently in Germany
Table 5. Risk ratio for fatal outcome for the STs linked to the serogroups significantly associated with death at 30 days after
diagnosis of IPD
For serogroup 1, MLST data were not known for 140 cases of IPD; for serogroup 3, MLST data were not known for 134 cases of IPD; for serogroup
11, MLST data were not known for 39 cases of IPD and for serogroup 19, MLST data were not known for 173 cases of IPD; therefore ‘All
non-missing’ indicates all cases of (serogroup 1, serogroup 3, serogroup 11 or serogroup 19) IPD with complete MLST data available for analysis.
Inf, infinity; RR, risk ratio; LCL, lower confidence limit; UCL, upper confidence limit.
Number Number diedPercentageRR LCL UCL
0.84 2.29 0.220.23
0.35 2.830.42 0.43
D. Inverarity and others
798Journal of Medical Microbiology 60
(Ru ¨ckinger et al., 2009), a study of 494 children identified
an overall case fatality rate of 5.3%. Serotype 7F had the
highest case fatality rate (14.8%) followed by serotype 23F
(8.3%) and serotype 3 (8.3%). In the Netherlands,
serotypes 3, 19F, 23A, 16F, 6B, 9N and 18C were also
recently associated with increased case fatality rates (Jansen
et al., 2009). The largest study assessing serotype asso-
ciation with death from 18858 patients with IPD was
published from Denmark and found (in patients over age 5
years) that serotypes 31, 11A, 35F, 17F, 3, 16F, 19F, 15B
and 10A were associated with higher mortality when
compared to serotype 1, but no associations with ST were
made (Harboe et al., 2009). The 30 day mortality overall
was 18% and in children under 5 years old it was 3%
(Harboe et al., 2009).
A prospective multi-centre study of 796 consecutive
patients from 10 countries (South Africa, USA, Sweden,
Spain, New Zealand, Taiwan, Argentina, Brazil, Hong
Kong and France) examined clinical outcome and mor-
tality at 14 days after the first positive blood culture for S.
pneumoniae (Alanee et al., 2007) and assessed for associa-
tions with particular serotypes categorized as invasive
(serotypes 1, 5 and 7), paediatric (serotypes 6, 9, 14, 19 and
23) and conjugate vaccine associated (serotypes 4, 6B, 9V,
14, 18C, 19F and 23F). This study focused predominantly
on adults, did not include patients from the UK and did
not look for associations between outcome and STs of
pneumococci. In fact, although it is recognized that
invasive capacity of the pneumococcus is dependent on
both serotype and genomic content (Garau & Calbo, 2007)
there is little published work which investigates whether
there is an association between pneumococcal ST or clonal
complex and disease outcome. Sjo ¨stro ¨m et al. (2006) per-
formed MLST on 105 pneumococcal isolates and related
these to disease severity by APACHE II score and case
fatality rate. Although only assessing between 3 and 41
isolates of individual STs, they did identify ST180 as having
a high case fatality rate (Sjo ¨stro ¨m et al., 2006).
We chose death or survival at 30 days after culture of
pneumococci from blood or CSF as our end point although
other similar studies have used 14 days (Alanee et al., 2007;
Yu et al., 2003). Up to 43% of deaths from pneumococcal
disease have been noted to occur in the first 24 h of
hospital admission (Austrian & Gold, 1964) and up to
64% of deaths occur within 5 days of hospital admission
(Ortqvist et al., 1993). As we did not access death certificate
records or patient notes for the primary cause of death we
cannot be certain that every death attributed to IPD was
directly caused by IPD but such a significant event within
30 days of death is likely to have been contributing to the
fatal outcome in the majority of cases.
It would be advantageous to perform this entire analysis
using serotypes rather than serogroups. However, during
the early years of the SHLMPRL strain collection and
database, investigation of pneumococcal serotyping using
factors for subtypes was not performed and so to be able to
utilize all the data available to us we performed this analysis
predominantly on serogroups. As serotypes 1 and 3 are
equivalent to serogroup 1 and 3 (as they have no subtypes)
this does not influence our findings for these serogroups.
In the years since 2003, serotyping data are complete and
so the data have been used to investigate whether
associations can be identified with fatal outcome for
individual serotypes, such as serotype 7F, which have
previously been associated with high case fatality rates
(Ru ¨ckinger et al., 2009).
Pneumococcal conjugate vaccination with a 7-valent
vaccine which protects against serotypes 4, 6B, 9V, 14,
18C, 19F and 23F (Prevnar, Wyeth) was introduced in
Scotland in September 2006 for infants. It is unlikely that
this will have influenced our results substantially as
mortality rates in the under 2 years age group (who would
be directly affected by the introduction of conjugate
vaccination) were low prior to vaccine introduction and
the serotypes covered are not associated with greater risk of
death in our analysis. From winter 2003, the 23-valent
polysaccharide vaccine (Pneumovax II, Aventis Pasteur)
was recommended to those aged 65 years and older in
Scotland. Analysis of the impact of the first year of this
intervention estimated 65% uptake of vaccine but did not
identify any significant impact on mortality due to IPD
(Mooney et al., 2008).
In this analysis, we were unable to look for associations
between particular serogroups or STs and social depriva-
tion scores or patient co-morbidities as such data are not
submitted to SHLMPRL nor can they be extrapolated from
other databases using the rudimentary epidemiological
information which is collected. Such associations are
highlighted in the study by Alanee et al. (2007), where
age over 65 years, underlying chronic disease, immuno-
suppression and severity of illness were identified as
independent risk factors significantly associated with
disease mortality. Even so, in Scotland, effects of social
deprivation on the incidence of IPD and the effect of
underlying medical conditions on case fatality rates and the
incidence of IPD have been previously documented (Kyaw
et al., 2003). Cases which featured in that analysis also
feature in this analysis of outcome although the effects of
variables other than age cannot be accounted for in our
analysis. Chen et al. (2009) in Taiwan also assessed the role
of comorbidities on outcome through multivariate analysis
in their study of children with IPD (which included
complicated pneumonia and meningitis) and found that
penicillin resistance (MIC¢2 mg ml21) was associated with
mortality as an independent risk factor. Penicillin resist-
ance in pneumococci was not shown to be associated with
death in the analysis by Yu et al. (2003), which assessed
clinical outcome of hospitalized adults with blood cultures
which were positive with growth of S. pneumoniae.
Between 1999 and 2007 only 7 of 4727 pneumococcal
isolates causing IPD in Scotland were fully penicillin
resistant (Cooke et al., 2010). Although it is now
recognized that penicillin breakpoints should be set lower
Risk of death from pneumococcal serogroups and STs
in cases of meningitis than in non-meningitis cases (for
instance the European Committee on Antimicrobial
Susceptibility Testing advise reporting of penicillin resis-
tance in cases of meningitis if the MIC is .0.064 mg ml21),
our previous analysis identified only 6 of 171 CSF isolates
with MICs between 0.12 and 1 mg ml21and none with an
MIC¢2 mg ml21(Cooke et al., 2010); therefore we do not
consider penicillin resistance to be substantially influencing
mortality from pneumococcal meningitis in the Scottish
population, as the vast majority of CSF isolates are fully
susceptible to penicillin. In bacteraemic pneumococcal
pneumonia, some evidence suggests improved outcome
when treatment involves use of a macrolide (Martı ´nez
et al., 2003; Metersky et al., 2007; Weiss et al., 2004) or
combination antibiotic therapy (Baddour et al., 2004;
Waterer et al., 2001) although other investigators have
found no association between initial antibiotic choice and
outcome (Aspa et al., 2006).
Unfortunately the retrospective nature of this study and the
lack of access to patients’ clinical notes and data regarding
antibiotic prescribing mean that it is impossible for us to
account for confounding by recognized independent risk
factors for mortality. Even so, it is also worth reiterating
the observation of Garau & Calbo (2007) that although the
above-noted independent risk factors are important in
determining outcome of IPD, there are substantial
numbers of patients with IPD who fit a category of young
adult with no pre-existing comorbidities, ‘in whom the
infecting serotype becomes the determinant factor of
It is of note that ST306 had a significant P-value before
accounting for age with the Cochran–Mantel–Haenszel
test analysis. ST306 [predominantly the Pneumococcal
Molecular Epidemiology Network (PMEN) clone Sweden1
ST306] has now become the commonest ST in Scotland
(Jefferies et al., 2010; Cooke et al., 2010; Lamb et al., 2008).
As further cases occur due to ST306 it may be that the P-
value for this ST reaches significance after accounting for
age. The possibility exists that ST306 may be associated with
reduced probability of death but that at present we are
unable to clearly demonstrate such an association. ST306 is
Scotland (Jefferies et al., 2010; Lamb et al., 2008). It is
interesting that no deaths in children due to serotype 1 were
identified in studies from Germany (Ru ¨ckinger et al., 2009),
in keeping with the hypothesis that serotype 1 is associated
with milder disease (Sjo ¨stro ¨m et al., 2006). It is known that
different pneumococcal serotypes produce different inflam-
matory responses in animal models, influencing disease
outcome (Engelhard et al., 1997; Mizrachi-Nebenzahl et al.,
2003). Interestingly, ST306 and ST191 have been found to
induce a low tumour necrosis factor (TNF) response and be
effectively cleared from the bloodstream of infected mice
(Sandgren et al., 2005). ST306 has been found not to cause
lethal murine disease (Sandgren et al., 2005). Our results
would be consistent with this also being possible in a human
population. The association that ST191 may have a reduced
risk of death is consistent with the findings of Sjo ¨stro ¨m et al.
(2006), who on assessing 34 ST191 isolates found a case
fatality rate of zero for this ST.
Although serotype 3 is associated with an increased relative
risk of death from IPD in Danish adults (Harboe et al.,
2009; Martens et al., 2004), in children, serotype 3-
associated ST180 pneumococci have been identified as
having an odds of invasiveness which was significantly
associated with asymptomatic carriage (Brueggemann
et al., 2003). Recently in Germany, serotype 3-associated
IPD has been shown to also have one of the highest case
fatality rates in children (Ru ¨ckinger et al., 2009). The
seemingly contradictory finding that serotype 3 pneumo-
cocci can cause disease with a high associated mortality in
some individuals while being harmlessly carried in the
nasopharynx of others has been recognized since the early
20th century (Blake, 1931). An association between
serotype 3 pneumococci causing invasive and severe disease
more commonly in the elderly than in children is also an
established observation (Blake, 1931; Cecil et al., 1927)
which remains true in several countries (Guimbao Besco ´s
et al., 2003; Inostroza et al., 2001; Kyaw et al., 2003; Martin
& Brett, 1996; Rahav et al., 1997; Shapiro & Austrian,
1994). It is therefore not surprising that serotype 3 has been
shown in this analysis to be significantly associated with a
fatal outcome even when age is taken into account. It is
interesting that ST180 was associated with fatal outcomes
(Fisher’s exact test) before accounting for age (Cochran–
Mantel–Haenszel test). Sjo ¨stro ¨m et al. (2006) also iden-
tified ST180 as having a high case fatality rate. This may be
a consequence of a strong association between ST180 and
serotype 3. ST180 has been associated with a serotype 19F
capsule in Germany and non-typable isolates in South
Korea but globally is predominantly associated with the
serotype 3 capsule.
We conclude from this analysis that there is a stronger
association between a fatal outcome and pneumococcal
capsular serogroup than there is between fatal outcome and
multilocus ST. As the over-75 age group has the highest
death rates associated with IPD and the implicated
serogroups are included in the 23-valent polysaccharide
vaccine (Pneumovax II, Sanofi Pasteur) which is offered to
all aged over 65 years in Scotland, there may be benefit in
further promotion of pneumococcal vaccination in the
over 65 age group, although there is debate over the
effectiveness of this vaccine (Huss et al., 2009). These
results also have an application in determining future
pneumococcal vaccine formulations.
More recent higher-valency conjugate vaccine formulations
which include serotype 3 may have an effect in reducing
deaths from IPD in infants (and possibly in adults through
herd immunity) while the introduction of serotype 1 into
conjugate vaccine formulations may reduce morbidity
from IPD but may have less effect on mortality from IPD.
D. Inverarity and others
800Journal of Medical Microbiology 60
We thank Mrs Joan Brown of the General Register Office for Scotland
for arranging database matching and assisting in the identification of
patients with IPD in Scotland who had died. We also acknowledge the
work of the late Dr Les Smart (Clinical Scientist) who was
instrumental in developing and implementing serotyping of pneu-
mococcal isolates in Scotland from 1992 until his death in 1997 at
what has since become the Scottish Haemophilus, Legionella,
Meningococcal and Pneumococcal Reference Laboratory (SHLMPRL).
This work utilized the Streptococcus pneumoniae MLST database
(http://spneumoniae.mlst.net/) hosted by Imperial College. This
work was presented in part at the 6th International Symposium on
Pneumococci and Pneumococcal Diseases, Reykjavik, Iceland, 8–12
K.L. was funded through an EPSRC CASE studentship with Wyeth
Pharmaceuticals. C.R. has received research funding from and has
acted as a consultant for Wyeth Pharmaceuticals. S.C.C. currently
receives unrestricted research funding from Pfizer Vaccines (pre-
viously Wyeth Vaccines). J.M.C.J. and S.C.C. have received
consulting fees from GlaxoSmithKline. S.C.C. and J.M.C.J. have
received financial assistance from vaccine manufacturers to attend
conferences. All grants and honoraria are paid into accounts within
the respective NHS Trusts or Universities, or to independent charities.
M.D. has received consulting fees from Novartis and has received
financial assistance from vaccine manufacturers to attend conferences.
M.D. has also received research funding from Sanofi Pasteur.
G.F.S.E. has received financial assistance from Wyeth Vaccines to
attend conferences. J.M.C.J., A.S., S.C.C., T.J.M., G.F.S.E. and
J.M. were co-recipients of research funding from Wyeth Vaccines.
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