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Bacterial meningitis in adults: Host and pathogen factors, treatment and outcome

Heckenberg, S.G.B.

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Citation for published version (APA):Heckenberg, S. G. B. (2013). Bacterial meningitis in adults: Host and pathogen factors, treatment and outcome.

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Bacterial m

enin

gitis in

adu

lts Sebastiaan

G.B

. Hecken

berg

Bacterial meningitis in adults

Host and pathogen factors, treatment and outcome

Sebastiaan G.B. Heckenberg

24407 Heckenberg_omslag 24-02-13 15:36 Pagina 1


host and pathogen factors,

treatment and outcome


24407 Heckenberg.indd 1 25-02-13 11:11

Colofon© 2013 S.G.B. Heckenberg

ISBN: 978-90-6464-650-8

Vormgeving: Ferdinand van Nispen, Citroenvlinder-dtp.nl, Bilthoven, the

Netherlands

Druk: GVO | Ponsen & Looijen, Ede, the Netherlands

24407 Heckenberg.indd 2 25-02-13 11:11

Bacterial meningitis in adults: host and pathogen factors,

treatment and outcome.

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof.dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op vrijdag 12 april 2013, te 14.00 uur

door

Sebastian Gerard Bartholomew Heckenberg

geboren te Amsterdam

24407 Heckenberg.indd 3 25-02-13 11:11

Promotiecommissie:

Promotor: Prof.dr. D. van de Beek

Overige leden: Prof. dr. P.W.M. Hermans

Prof. dr. T. van der Poll

Prof. dr. P. Portegies

Prof. dr. I.N. van Schaik

Prof. dr. B.M.J. Uitdehaag

Faculteit der Geneeskunde

24407 Heckenberg.indd 4 25-02-13 11:11

ContentsChapter 1 Introduction 7

Chapter 2 Clinical features, outcome and meningococcal

genotype in 258 adults with meningococcal meningitis

13

Chapter 3 Naturally occurring lipid A mutants in Neisseria

meningitidis from patients with invasive meningococcal

disease are associated with reduced coagulopathy

31

Chapter 4 Dexamethasone in adults with meningococcal

meningitis

61

Chapter 5 Nationwide evaluation of implementation and

effectiveness of adjunctive dexamethasone in adult

pneumococcal meningitis

75

Chapter 6 Hearing loss in adults surviving pneumococcal

meningitis is associated with otitis and pneumococcal

serotype

95

Chapter 7 Complement component 5 contributes to poor disease

outcome in humans and mice with pneumococcal

meningitis

109

Chapter 8 General discussion: bacterial meningitis: epidemiology,

pathophysiology and treatment

145

Summary 175

Samenvatting 187

Dankwoord 191

CV 191

List of publications 195

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Chapter 1

Introduction

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Chapter 1

8

Bacterial meningitis occurs when bacteria invade the subarachnoid space

surrounding the brain and spinal cord. This infection and the ensuing

inflammatory response cause severe, life-threatening disease. Until the

advent of antibacterial agents effective treatment was lacking. After initial

success using intrathecal meningococcal antiserum in the first decades of the

twentieth century, the introduction of sulfonamides in the 1930s provided the

first effective antibiotic therapy for bacterial meningitis caused by Haemophilus

influenzae type B and Neisseria meningitidis. Subsequently, penicillin treatment

for pneumococcal meningitis was implemented in the 1940s. In meningococcal

disease, increasing resistance to sulfonamides prompted its replacement by

penicillin. 1, 2

Epidemiology

To monitor the epidemiology of bacterial meningitis, the Netherlands Reference

Laboratory for Bacterial Meningitis was officially established in 1975 and

isolates from cerebrospinal fluid (CSF) from patients with bacterial meningitis

have been stored and collected. The implementation of conjugate vaccination

against type B H. influenzae, group C N. meningitidis, and most recently, the

pneumococcal vaccine have reduced the incidence of bacterial meningitis

in the Netherlands. Currently, approximately 85% percent of all bacterial

meningitis is caused by N. meningitidis and Streptococcus pneumoniae. Other

causes are Listeria monocytogenes, H. influenzae and Streptococcus agalactiae.3

N. meningitidis (the meningococcus) is a common inhabitant of the human

nasopharynx. Carriage is found exclusively in humans. Disease occurs when

meningococci invade the mucosal space and enter the bloodstream. Invasive

disease can progress swiftly and fatally, mainly through sepsis and meningitis.

The incidence of meningococcal meningitis in adults in the Netherlands is

approximately 1 per 100,000. In the Netherlands, the most common serogroups

are B and C, while serogoup A is the cause of severe epidemics in the “meningitis-

belt” in sub-Saharan Africa. Since the implementation of vaccination with the

MenC vaccine, the incidence of group C disease has decreased substantially in

the Netherlands. In 1998, a new method of typing meningococci was described

using nucleotide sequencing of meningococcal genes. Multilocus sequence

typing (MLST) has provided a useful tool for the unambiguous characterisation

24407 Heckenberg.indd 8 25-02-13 11:11

IntroductIon

9

Ch

apt

er 1of meningococci and allows for rapid identification of invasive lineages of

meningococci.4

S. pneumoniae (the pneumococcus) is a major cause of respiratory infections,

sepsis and meningitis worldwide. In pneumococcal meningitis, mortality

remains high (20-30%) despite effective antiobiotic treatment. Over 90

serotypes have been described. Increasing antibiotic resistence is an emerging

challenge worldwide with rates of resistance exceeding 50% in parts of the

United States. However, antibiotic resistance in the Netherlands remains low. In

2006, the heptavalent pneumococcal vaccine was implemented in the national

vaccination program in the Netherlands.

Treatment and outcome

Although the prognosis of patients with bacterial meningitis has improved

greatly through effective antibiotic treatment, substantial morbidity and

mortality has remained, fuelling research into adjunctive treatment. Following

experimental animal studies, attenuation of the severe inflammatory

response emerged as an important pathway for improving clinical outcome.

Since the 1960s, clinical trials with adjunctive corticosteroid treatment have

been performed with conflicting results. However, meta-analyses showed a

reduction in hearing loss in patients treated with adjunctive corticosteroids.5

In 2002, a European randomized clinical trial showed beneficial effect of

adjunctive dexamethasone in adults with bacterial meningitis. The effect was

most pronounced in pneumococcal meningitis and mortality in those patients

was reduced by 10%. However, these results were not reproduced in clinical

trials from other parts of the world. An individual patient data-analysis and

subsequent Cochrane review supported the continued use of dexamethasone

in children and adults in high-income countries. 6-8

Adequate antibiotic and adjunctive therapy in combination with supportive

care have reduced the mortality of bacterial meningitis, but neurologic

sequelae in patients surviving bacterial meningitis are common, particularly

in pneumococcal meningitis. They include cognitive impairment, hearing loss,

epilepsy and other focal neurological deficits. 9-11

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Chapter 1

10

Aims and outline of this thesis

In chapter 2 we present the results of a nationwide study in adults with

meningococcal meningitis. Details of clinical characteristics, therapy and

outcome are presented, as well as the correlation of bacterial genotype,

acquired through MLST analysis, and clinical characteristics. Chapter 3

describes the collaborative effort of our research group with the Netherlands

Vaccine Institute. The discovery of meningococci with an impaired potential

to induce cytokine production is described. Furthermore, the mutations in

bacterial genome are revealed and the relationship with clinical characteristics

in patients from our nationwide cohort studies is investigated. In chapters 4

and 5, we present the results of nationwide studies from 2006-2009 on the

implementation of dexamethasone treatment in the Netherlands. Outcome in

patients was compared to a cohort of 1998-2002, before the implementation

of dexamethasone. The influence of dexamethasone treatment on outcome in

meningococcal meningitis is described in chapter 4. Chapter 5 describes the

change in outcome in adults wih pneumococcal meningitis and we compare

the observed outcome with that in a prognostic model. Chapter 6 describes the

incidence of hearing loss following pneumococcal meningitis, combining two

nationwide studies of adults with bacterial meningitis, from 1998-2002 and from

2006-2009. The association between clinical characteristics, pneumococcal

serotype and occurrence of hearing loss is described. In Chapter 7 the results

of our cooperation with a research group in Munich, Germany are presented.

We describe the association of unfavorable outcome with a single nucleotide

polymorphism (SNP) coding for complement factor C5. Next, C5 fragment

levels in CSF and the relationship with clinical characteristics were investigated.

Finally, a mouse model of C5a deficient mice and adjuvant treatment with

C5-specific monoclonal antibodies are described. In chapter 8, we conclude

with a general discussion describing the epidemiology, pathophysiology and

treatment of bacterial meningitis incorporating the results of the presented

studies and suggestions for future research are proposed.

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IntroductIon

11

Ch

apt

er 1References

1. Uiterwijk A, Koehler PJ. A history of acute bacterial meningitis. J Hist Neurosci 2012;21(3):293-313.

2. Swartz MN. Bacterial meningitis - a view of the past 90 years. N Engl J Med 2004;351(18):1826-1828.

3. Tunkel AR. Bacterial Meningitis. Philadelphia: Lippincott, Williams & Wilkins; 2001.

4. Maiden MC, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 1998;95(6):3140-3145.

5. McIntyre PB, Berkey CS, King SM et al. Dexamethasone as adjunctive therapy in bacterial meningitis. A meta-analysis of randomized clinical trials since 1988. JAMA 1997;278(11):925-931.

6. Brouwer MC, McIntyre P, de Gans J, Prasad K, van de Beek D. Corticosteroids for acute bacterial meningitis. Cochrane Database Syst Rev 2010;9:CD004405.

7. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347(20):1549-1556.

8. van de Beek D, Farrar JJ, de Gans J et al. Adjunctive dexamethasone in bacterial meningitis: a meta-analysis of individual patient data. Lancet Neurol 2010;9(3):254-263.

9. Weisfelt M, van de Beek D, Spanjaard L, Reitsma JB, de Gans J. Clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series. Lancet Neurol 2006;5(2):123-129.

10. Ostergaard C, Konradsen HB, Samuelsson S. Clinical presentation and prognostic factors of Streptococcus pneumoniae meningitis according to the focus of infection. BMC Infect Dis 2005;5:93.

11. Zoons E, Weisfelt M, de Gans J et al. Seizures in adults with bacterial meningitis. Neurology 2008;70(22 Pt 2):2109-2115.

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Chapter 2

Clinical features, outcome, and meningococcal genotype

in 258 adults with meningococcal meningitis: a prospective cohort study


Jan de Gans

Matthijs C. Brouwer

Martijn Weisfelt

Jurgen R. Piet

Lodewijk Spanjaard

Arie van der Ende

Diederik van de Beek

Medicine (Baltimore), 2008;87(4):185-92

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Chapter 2

14

Abstract

Meningococcal meningitis remains a life-threatening disease. Neisseria

meningitidis is the leading cause of meningitis and septicemia in young adults

and is a major cause of endemic bacterial meningitis worldwide. The Meningitis

Cohort Study was a Dutch nationwide prospective observational cohort study

of adults with community-acquired bacterial meningitis, confirmed by culture

of cerebrospinal fluid, from October 1998 to April 2002. Patients underwent

a neurologic examination at discharge, and outcome was graded with the

Glasgow Outcome Scale. Serogrouping, multi locus sequence typing, and

susceptibility testing of meningococcal isolates were performed.

The study identified 258 episodes of meningococcal meningitis in 258 patients.

The prevalence of the classical triad of fever, neck stiffness, and change in

mental status was low (70/258, 27%). When rash was added to the classical

triad, 229 of 258 (89%) patients had at least 2 of 4 signs. Systolic hypotension

was associated with rash (22/23 vs. 137/222, p=0.002) and absence of neck

stiffness (6/23 vs. 21/220, p=0.05). Neuroimaging before lumbar puncture was

an important cause of delay of therapy: antibiotics were not initiated before

computed tomography (CT) scan in 85% of patients who underwent CT scan

before lumbar puncture. Unfavorable outcome occurred in 30 of 258 (12%)

patients, including a mortality rate of 7%. Neurologic sequelae occurred in 28

of 238 (12%) patients, particularly hearing loss (8%). Factors associated with

sepsis and infection with meningococci of clonal complex 11 (cc11) are related

with unfavorable outcome.

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Meningococcal Meningitis in adults

15

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Introduction

Bacterial meningitis is a life-threatening infectious disease. The estimated

incidence of bacterial meningitis is 2.6–6.0 cases per 100,000 adults per year in

developed countries and is up to 10 times higher in less developed countries.1

The predominant causative pathogens in adults are Streptococcus pneumoniae

(pneumococcus) and Neisseria meningitidis (meningococcus), which are

responsible for 80%–85% of all cases.2

The meningococcus is the leading cause of meningitis and septicemia in

young adults and is a major cause of endemic bacterial meningitis worldwide.3

The epidemiology of meningococcal disease is important for vaccination

strategies.3, 4 Whereas meningococci of serogroups B, C, and Y were responsible

for several recent outbreaks of invasive disease in the United States and other

developed countries, serogroup A meningococci are the primary cause of

endemic disease in developing countries.3 Current meningococcal vaccines

are based on the capsular polysaccharides of serogroup A, C, W-135, and

Y meningococci.4 Multilocus sequence typing (MLST) is considered the

gold standard for genotyping of meningococci and can be used to study

epidemiology.4, 5

We previously described a prospective cohort of 696 adult patients with

community-acquired bacterial meningitis.2 We now provide a detailed

description of clinical features, prognostic factors, outcome, and MLST in the

subset of 258 adults with meningococcal meningitis.

Patients and methods

The Dutch Meningitis Cohort Study was a prospective nationwide observational

cohort study of adults with community-acquired bacterial meningitis in the

Netherlands. Inclusion and exclusion criteria have been described extensively

elsewhere.2 In summary, eligible patients were older than 16 years, had bacterial

meningitis confirmed by culture of cerebrospinal fluid (CSF), and were listed in

the database of the Netherlands Reference Laboratory for Bacterial Meningitis

from October 1998 to April 2002. This laboratory receives CSF isolates from

about 85% of all patients with bacterial meningitis in the Netherlands. The

treating physician was contacted, and informed consent was obtained from

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Chapter 2

16

all participating patients or their legally authorized representatives. Compared

with the original dataset, 1 additional patient with meningococcal meningitis

was identified.2 This observational study with anonymous patient data was

carried out in accordance with the Dutch privacy legislation.

Procedures

Patients underwent a neurologic examination at discharge, and outcome

was graded with the Glasgow Outcome Scale. This measurement scale is well

validated with scores varying from 1 (indicating death) to 5 (good recovery). A

favorable outcome was defined as a score of 5, and an unfavorable outcome as

a score of 1–4. Focal neurologic abnormalities were divided into focal cerebral

deficits (aphasia, monoparesis, or hemiparesis) and cranial nerve palsies.

Whenever audiometry was done, hearing loss was classified as normal (<30

decibels [dB]), mild (30–55 dB), moderate (55–70 dB), severe (70–90 dB), or

profound (>90 dB).6 Patients using immunosuppressive drugs and those with

diabetes mellitus, alcoholism, asplenia, or human immunodeficiency virus

(HIV) infection were considered to be immunocompromised.

Causes of death were independently classified in 2 categories by 2 clinicians

as described previously.7 The 2 categories were 1) systemic causes, including

septic shock, respiratory failure, multiple-organ dysfunction, cardiac ischemia;

and 2) neurologic causes, including brain herniation, cerebrovascular

complications, intractable seizures, and withdrawal of care because of poor

neurologic prognosis. Only patients who died within 14 days after admission

were classified, because death within this period is probably caused by direct

consequences of the meningitis.8 Differences in classification between the 2

clinicians were resolved by discussion.

Serogrouping, MLST, and susceptibility testing of meningococcal isolates were

performed by the Netherlands Reference Laboratory for Bacterial Meningitis.

Serogrouping and penicillin-susceptibility testing were performed as described

elsewhere.2 MLST was performed on all available strains (n = 254) as described

by Maiden et al;5 4 meningococcal strains were not available for analyses. MLST

uses sequence data obtained from 7 housekeeping genes.5 The alleles from

these housekeeping genes are assigned allele numbers, and the combination

of these allele numbers makes up a sequence type. Clonal complexes were

allocated according to the online MLST-database.9

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Statistical Analysis

The Mann-Whitney U test was used to identify differences between groups in

continuous variables, and dichotomous variables were compared by the chi-

square or Fisher exact test. We did not perform multivariate analysis since the

number of patients with unfavorable outcome was limited. All statistical tests

were 2-tailed, and a p value less than 0.05 was regarded as significant.

Results

A total of 258 episodes of meningococcal meningitis occurred in 258 patients; in

1 patient CSF cultures yielded both N. meningitidis and group B streptococcus.

The calculated annual incidence of community-acquired meningococcal

meningitis was approximately 1 case per 100,000 adults.

Patient characteristics are presented in Table 1. The median age was 36 years

(interquartile range [IQR], 19–50 yr), and 52% were male. Two patients had

a family history of bacterial meningitis. Otitis or sinusitis was present in 9 of

258 (4%) patients, and pneumonia in 13 of 258 (5%) patients. Seizures before

admission were present in only 2 of 255 patients. Acute onset of illness, defined

as duration of symptoms before admission less than 24 hours, was present in

123 of 251 (49%) patients. Six patients were treated with antibiotics before

presentation in the emergency department (4 orally, 2 intravenously).

The prevalence of the triad of fever, neck stiffness, and change in mental status

was low (27%; see Table 1). Relatively small proportions of patients had fever

(temperature >38.0 8C, 64%) or change in mental status (defined as a score

on the Glasgow Coma Scale (GCS) <14; 51%); 246 of 258 (95%) patients had at

least 2 of 4 signs (classic triad plus headache). Focal neurologic abnormalities

were present in 56 of 256 (22%) patients, including aphasia in 29 of 256 (11%)

patients. Cranial nerve palsy was present in 18 of 256 (7%) patients; N.III in 1

patient, N.VI in 6, N.VII in 3, N.VIII in 9 patients; 1 patient had a palsy of both N.VI

and N.VIII.

At presentation, signs of septic shock (defined as diastolic blood pressure <60

mm Hg, systolic blood pressure ≤90 mm Hg and/or heart rate ≥120/min) were

present in 74 of 241 (31%) evaluated patients. Rash was present in 164 (64%)

patients and was characterized as petechial in 145 of 160 (91%) patients; 41

also had purpura and/or ecchymoses. In 15 of 160 (9%) patients, only purpura

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Chapter 2

18

or ecchymoses were noted; rash was not specified in 4 patients. Systolic

hypotension was associated with presence of rash (22/23 [96%] vs. 137/222

[62%], p=0.002) and absence of neck stiffness (6/23 [26%] vs. 21/220 [10%],

p=0.05). When rash was added to the classical triad of fever, neck stiffness, and

change in mental status, 229 of 258 (89%) patients had at least 2 of 4 signs. Of

the 12 patients without any signs of the classical triad plus headache, 10 (83%)

did present with rash.

Lumbar puncture was performed in all patients. Opening pressures were

measured with a water-manometer in 76 of 258 (29%) patients. The median

pressure was 36 cm water (IQR, 22–50 cm); very high pressures (>40 cm

water) were found in 33 (43%) patients. In 214 of 248 (86%) patients, at least 1

individual CSF finding predictive of bacterial meningitis was present (glucose

concentration <1.9 mmol/L, ratio of CSF glucose to blood glucose <0.23,

protein concentration >2.20 g/L, CSF white blood cell count [WBCC] >2000/

mm3, or CSF neutrophil count >1180/mm3).10 A CSF WBCC of <1000/mm3 was

found in 47/242 (19%) patients; in these patients, systolic hypotension was

more common (12/42 vs. 8/188, p<0.0001).

Five patients had a normal initial CSF analysis (defined as CSF WBCC ≤5/mm3,

CSF protein ≤0.50 g/L, and ratio of CSF glucose to blood glucose ≥0.40); all

of these 5 patients presented with rash, and Gram staining of CSF showed

bacteria in 3. Gram staining of CSF was done for 244 of 258 (95%) patients

and showed bacteria in 216 (89%) patients. Findings of Gram staining were

indicative of N. meningitidis in 209 of 244 (86%) patients. In 5 patients, findings

were interpreted as pneumococci; all of these patients presented without rash.

Routine blood examination was performed in all patients. To explore indexes of

inflammation in CSF and blood we performed an analysis of correlations. There

was a significant correlation between low WBCC in CSF and blood (Spearman r

0.28, p<0.0001). Low CSF WBCC was also significantly associated with low CSF

protein level (0.52, p<0.0001), low blood thrombocyte count (0.19, p=0.004),

and lower indexes of inflammation in the blood: erythrocyte sedimentation rate

(ESR) (0.16, p=0.03), and C-reactive protein (CRP) (0.40, p<0.0001). Blood WBCC

was significantly associated with blood thrombocyte count (0.34, p<0.0001),

but not with ESR (0.12, p=0.10) or CRP (0.15, p=0.08).

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Table 1. Clinical and laboratory characteristics of 258 episodes of meningococcal meningitis

Characteristic

Demographics

Age – yr (mean±SD) 36±19

Male sex 133 (52)

Duration of symptoms <24 hr 123/251 (49)

Pretreated with antibiotics 6/257 (2)

Pneumonia 13/258 (5)

Immunocompromised 10/258 (4)

Symptoms and signs at presentation

Headache 223/247 (90)

Nausea 194/247 (79)

Neck stiffness 226/255 (89)

Rash 164/256 (64)

Systolic blood pressure (BP)– mmHg 125 (110-140)

Diastolic BP – mmHg 71 (60-80)

Diastolic BP <60 mmHg 37/246 (15)

Heart rate – beats/minute 95 (80-110)

Fever (T ≥38.0 ºC) 161/250 (64)

Impaired consciousness (GCS <14) 131/257 (51)

Coma (GCS <8) 19/257 (7)

Focal neurologic deficits

Cranial nerve palsy 18/258 (7)

Cerebral palsy 32/258 (12)

Triad of fever, neck stiffness and change in mental status 70/258 (27)

Cerebrospinal fluid parameters

Opening pressure – cm of water 40 (22-50)

White cell count – cells/mm³ 5328 (1590-12433)

<100 cells/mm³ 21/242 (9)

100-999 cells/mm³ 26/242 (11)

>999 cells/mm³ 195/242 (80)

Protein – g/L 4.5 (2.2-7.0)

CSF/Blood glucose ratio 0.08 (0.01-0.30)

Blood parameters

Positive blood culture 129/227 (57)

Erythrocyte sedimentation rate (ESR) – mm/hr 40±38

C-reactive protein – mg/L 240±114

Sodium – mmol/L 137±4

Thrombocyte count – 109/L 180±89

Creatinin – μmol / L 113±61

Data are number/number assessed (%) or median (IQR) unless otherwise stated. Heart rate was evaluated in 240 episodes. Opening pressure was evaluated in 76 episodes. White cell count was evaluated in 242 episodes. CSF Protein was evaluated in 238 episodes. CSF/Blood glucose ratio was evaluated in 230 episodes. ESR was evaluated in 200 episodes. C-reactive protein was evaluated in 150 episodes. Sodium was evaluated in 255 episodes. Thrombocyte count was evaluated in 245 episodes. Creatinin was evaluated in 251 episodes.

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Chapter 2

20

Neuroimaging was performed in 133 of 258 (52%) patients and consisted of

computed tomography (CT) in all patients. Cranial CT was done on presentation

in 92 of 133 (69%) patients. CT abnormalities were found in 15 of these patients

and consisted of cerebral edema in 8 (9%) and signs of hydrocephalus in 4 (4%).

Neuroimaging preceded lumbar puncture in 85 of 92 (92%) patients; antibiotics

were administered before CT in 15 of 88 (17%) of these patients. Indications for

performing CT before lumbar puncture (defined as a score on the GCS <10,

focal cerebral deficits, new onset seizures, or papilledema) were present in 90

of 258 (35%) patients. Of these 90 patients who met these criteria of CT before

lumbar puncture, 44 (49%) underwent CT before lumbar puncture. During

the clinical course, cranial CT was performed in 41 additional patients. CT was

prompted by a decline in level of consciousness in 10 patients, focal neurologic

abnormalities in 14 patients, and persistent fever in 11 patients. Cerebral

edema was found in 4 patients and subdural empyema, slight hydrocephalus

and multiple abscesses were found in 1 scan each. Other scans were reported

as normal.

Initial antimicrobial treatment consisted of monotherapy penicillin or

amoxicillin in 92 (36%), monotherapy third-generation cephalosporin in 21

(8%), penicillin or amoxicillin plus third-generation cephalosporin in 77 (30%),

and another regimen in 63 of 253 (25%) patients. Adjunctive steroid treatment

was administered to 43 patients; the regimen was specified in 37 of those. In 12

of 37 patients, 10 mg of dexamethasone was administered before or with the

first dose of antibiotics and given every 6 hours for 4 days. In the remaining 25

patients, various steroid regimens were administered after antibiotic treatment

had started. Steroids used were hydrocortisone, dexamethasone, prednisone,

and prednisolone; the median daily dose equivalent to dexamethasone was 15

mg (range, 3–100 mg), and duration of treatment varied between 1 and 7 days.

During the clinical course, neurologic or systemic complications developed

in 113 of 258 (44%) patients. Cardiorespiratory failure occurred in 44 of 258

(17%) patients, requiring mechanical ventilation in 35. Patients who developed

cardiorespiratory failure during the clinical course were more likely to have

systolic hypotension on admission (18/23 [78%] vs. 22/223 [10%], p<0.0001),

CSF WBCC <1000/mm3 (21/41 [51%] vs. 26/201 [13%], p<0.0001), and rash (40/44

[91%] vs. 124/212 [58%], p<0.0001). One or more neurologic complications

(impairment of consciousness, seizures, or focal neurologic abnormalities)

developed in 105 of 258 (41%) patients.

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CSF culture yielded N. meningitidis in all patients. Blood samples were cultured

in 227 patients, of which 129 (57%) were positive. Positive blood cultures

were related with presence of rash (103/128 [80%] vs. 48/98 [49%], p<0.0001).

Antibiotic susceptibility was tested in 256 strains; 252 strains were sensitive to

penicillin, and 4 strains showed intermediate susceptibility to penicillin. Initial

antimicrobial therapy was judged to be microbiologically adequate in 249

of 253 (98%) patients. All 4 patients with inadequate therapy were infected

with intermediately susceptible strains and were treated with monotherapy

amoxicillin or penicillin; outcome was favorable in all 4 patients. Antimicrobial

therapy was stepped down to monotherapy penicillin or amoxicillin within 3

days in 179 of 253 (71%) cases.

The serogrouping result was available for 256 meningococcal strains. Of these,

173 (68%) were of serogroup B, 79 (31%) of serogroup C, 3 (1%) of serogroup Y,

and 1 (<1%) was of serogroup W135. Of 258 isolates, 254 (98%) were analyzed

by MLST (Table 2). MLST analysis of 254 isolates showed 91 unique sequence

types. The most prevalent clonal complexes (cc) were cc41/44 (41%), cc11

(24%), and cc32 (16%). All cc11 strains were serogroup C. During the study

period there was an increase in disease caused by meningococci belonging to

cc11 (Figure 1).

In a univariate analysis we explored relations between different clonal

complexes and clinical characteristics. Table 3 represents univariate testing

characteristics of infection by meningococci of 1 clonal complex versus

infection by meningococci of all other complexes. Results should be interpreted

with caution because of the explorative nature and multiple relations tested.

The main finding of this exploration is the relation between infection by

meningococci of cc11, disease severity, and characteristics related with sepsis

(low level of consciousness, p=0.02; CSF WBCC <1000/mm3, p=0.01; low blood

WBCC, p=0.03; high serum creatinine, p=0.003; positive blood culture, p=0.03).

Blood pressure was not related with clonal complex.

Of 258 patients, 19 (7%) died; 13 of the 19 (68%) within 24 hours. Sepsis was the

leading cause of mortality (14 of 19 fatalities, 73%); 3 additional patients died of

systemic causes (cardiorespiratory failure in 2 patients and multi-organ failure

in 1 patient). At discharge, neurologic examination was performed in 238 of

239 (99%) patients and revealed focal neurologic abnormalities in 28 (12%);

hearing loss was the most common neurologic sequelae in 19 (8%) (Table 4).

Hearing loss appeared after a median of 2 days (range, 0–24 d), and results of

24407 Heckenberg.indd 21 25-02-13 11:11

Chapter 2

22

audiometry were available for 9 patients. Hearing loss was unilateral in 3 (33%)

and bilateral in 6 (67%) patients. The severity of hearing loss was classified as

profound in 3 (33%), severe in 1 (11%), moderate in 1 (11%), mild in 3 (33%),

and normal in 1 (11%).6 Outcome was graded as favorable in 228 (88%) and

unfavorable in 30 (12%) patients (see Table 4).

Table 2. Frequency of clonal complexes and serogroups

Clonal complex No (%) Serogroup B Serogroup C Serogroup Y

cc41/44 104 (41) 103 1 -

cc11 62 (24) - 62 -

cc32 41 (16) 41 - -

cc269 16 (6) 14 2 -

cc8 10 (4) - 10 -

cc60 4 (2) 3 1 -

cc461 2 (1) 1 1 -

cc18 2 (1) 1 1 -

Othera 7 (3) 3 - 3

No ccb 6 (2) 6 - -

Total 254 172 78 3acc35, cc167, cc213, cc1286 (serogroup W135), cc3544, cc5457, cc5453. b ST-212, ST-2700, ST-3549, ST 3621, ST-4258, ST-5408.

Figure 1. Number of cases due to meningococci of cc11 during study period

jan-99 jul-99 jan-00 jul-00 jan-01 jul-01 jan-02Time

0

5

10

15

20

sesac fo rebmu

N

24407 Heckenberg.indd 22 26-02-13 09:17


23

Ch

apt

er 2

Tab

le 3

. Clo

nal c

omp

lexe

s an

d cl

inic

al c

hara

cter

istic

s

cc41

/44

N=

104

cc11

N=

62cc

32N

=41

NP

NP

NP

Dur

atio

n of

sym

pto

ms

<

24 h

r52

/101

(52)

-.5

831

/61(

51)

-.8

017

/40

(43)

-.3

4

Rash

69

/103

(67)

-.4

640

/61

(66)

-.8

123

/41

(56)

-.2

3

Syst

olic

blo

od p

ress

ure

<

90 m

mH

g 7/

98 (7

)-

.39

8/60

(13)

-.4

44/

40 (1

0)-

.80

Dia

stol

ic b

lood

pre

ssur

e <

60

mm

Hg

13/9

8 (1

3)-

.48

12/6

0 (2

0)-

.24

6/40

(15)

-.9

7

Hea

rt ra

te

>12

0 b

eats

/min

ute

17/9

6 (1

6)-

.86

9/57

(16)

-.8

38/

40 (2

0)-

.43

Gla

sgow

Com

a Sc

ale

scor

e 12

±3

104

.55

11±

361

.02

13±

340

.25

Com

a (G

CS

<8)

6/

104

(6)

-.3

87/

61 (1

2)-

.18

1/40

(2)

-.1

8

Cra

nial

ner

ve p

alsy

18

/104

(17)

-.5

914

/62

(23)

-.3

93/

41 (7

)-

.04

Cer

ebra

l pal

sy

9/10

4 (9

)-

.12

12/6

2 (1

9)-

.07

2/41

(5)

-.1

0

CSF

whi

te c

ell c

ount

-

10³c

ells

/mm

³ 11

.6±

19.8

100

.12

10.2

±17

.359

.11

17.3

±61

.536

.70

CSF

whi

te c

ell c

ount

<

1000

cel

ls/m

m³

16/1

00 (1

6)-

.22

19/5

9 (3

2)-

.01

5/36

(14)

-.3

4

ESR

– m

m/h

r 38

±39

87.6

436

±28

42.8

646

±35

31.2

8

Bloo

d w

hite

cel

ls -

109 /L

22

±9

104

.04

18±

1061

.03

20±

741

.80

Thro

mb

ocyt

e co

unt –

p

late

lets

/mm

³ 17

8±77

99.6

317

7±10

959

.40

185±

8840

.82

Cre

atin

in -

μmol

/ L

111±

5710

1.6

413

8±80

61.0

097

±52

40.0

9

Posi

tive

blo

od c

ultu

re

48/8

8 (5

5)-

.34

39/5

9 (6

6)-

.03

20/3

4 (5

9)-

.57

Favo

rab

le o

utco

me

(GO

S 5)

94

/104

(90)

-.3

749

/62

(79)

-.0

137

/41

(90)

-.6

6

cc26

9n=

16cc

8n=

10O

ther

ST

n=21

nP

nP

NP

7/15

(47)

-.8

32/

9 (2

2)-

.10

13/2

1 (8

1)-

.23

15/1

6 (9

4)-

.01

8/10

(80)

-.2

97/

21 (3

3)-

.00

2/16

(13)

-.6

21/

9 (1

1)-

.66

1/20

(5)

-.7

7

2/16

(13)

-.7

52/

9 (2

2)-

.55

2/20

(10)

-.5

0

3/16

(19)

-.5

41/

9 (1

1)-

.81

3/19

(16)

-.8

5

12±

416

.87

12±

310

.95

13±

321

.35

3/16

(19)

-.0

80/

10 (0

)-

.36

2/21

(10)

-.7

2

2/16

(13)

-.5

05/

10 (5

0)-

.01

6/21

(29)

-.2

4

2/16

(13)

-.9

92/

10 (2

0)-

.47

5/21

(24)

-.1

1

11.5

±23

.615

.80

9.8±

9.6

10.7

37.

2±5.

518

.84

3/15

(20)

-.9

81/

10 (1

0)-

.43

3/18

(17)

-.7

3

34±

2614

.67

33±

2610

.56

59±

7812

.52

22±

916

.32

14±

910

.03

21±

920

.96

203±

114

15.5

717

6±89

10.6

817

2±63

18.9

5

96±

3216

.49

98±

2810

.77

101±

4919

.41

7/14

(50)

-.8

57/

10 (7

0)-

.33

7/18

(39)

-.2

5

15/1

6 (9

4)-

.48

9/10

(90)

-.8

620

/21

(95)

-.3

0

Dat

a ar

e nu

mb

er/n

umb

er a

sses

sed

(%) o

r mea

n±SD

.

24407 Heckenberg.indd 23 25-02-13 11:11

Chapter 2

24

Table 4. Outcome

Outcome No./No. assessed (%)

Neurological deficits at discharge

Hearing loss (Eighth cranial nerve palsy)

19/237 (8)

Other cranial nerve palsy 6/238 (3)

Aphasia 1/238 (0.4)

Monoparesis 1/238 (0.4)

Hemiparesis 2/238 (1)

Quadriparesis 2/238 (1)

Glasgow outcome scale

1 (death) 19/258 (7)

2 (vegetative state) 0/258 (0)

3 (severe disability) 4/258 (2)

4 (moderate disability) 7/258 (3)

5 (mild/no disability) 228/258 (88)

Factors associated with unfavorable outcome (Table 5) were advanced age,

absence of neck stiffness, presence of rash, systolic or diastolic hypotension,

tachycardia, low CSF WBCC, low CSF protein level, high CSF/blood glucose

ratio, positive blood culture, high serum creatinine level, and low level of

thrombocytes. The proportion of patients who died was significantly higher

in patients without a CSF predictor for meningitis than in those with at least

1 CSF finding considered predictive for bacterial meningitis (10/33 [30%]

vs. 8/214 [4%], p<0.0001). Risk for unfavorable outcome was significantly

higher in patients infected by meningococci of cc11 (all of which were group

C meningococci) compared to patients infected by meningococci of other

clonal complexes (13/62 [21%] vs. 17/192 [9%], p=0.01). Although serogroup

was not significantly associated with outcome in univariate analysis, the risk

for an unfavorable outcome tended to be higher in patients with serogroup C

disease, compared with those with non-serogroup C disease (14/79 [18%] vs.

16/177 [9%], p=0.06).

24407 Heckenberg.indd 24 25-02-13 11:11


25

Ch

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Table 5. Univariate analysis of predictors of unfavorable outcome in 258 episodes of meningococcal meningitis

Unfavorable Favorable p

Demographics

Age , yr (mean ±SD) 46±22 35±18 .13

Duration of symptoms <24 hr 16/30 (53) 107/221 (48) .61

Symptoms at presentation

Neck stiffness 22/29 (76) 204/226 (90) .02

Rash 25/30 (83) 139/226 (61) .02

Systolic blood pressure <90 mmHg 12/28 (43) 11/218 (5) <.0001

Diastolic Blood Pressure <60 mmHg 12/28 (43) 25/218 (11) <.0001

Heart rate >120 bpm 11/28 (40) 30/212 (14) .004

Fever (T ≥38.0º C) 19/28 (68) 142/222 (64) .69

Impaired consciousness (GCS Score <14) 19/30 (63) 112/227 (49) .15

Coma (GCS <8) 4/30 (13) 15/227 (7) .19


- Cranial nerve palsy 8/30 (27) 40/228 (18) .23

- Cerebral palsy 5/30 (17) 27/228 (12) .45

Cerebrospinal fluid parameters

Opening pressure – cm of water 34 (14-50) 40 (24-40) .58

White cell count <1000/mm3 – no. (%) 15/27 (56) 32/215 (15) <.0001

Protein – g/L 2.9 (0.6-4.9) 4.9 (2.4-7.3) .006

CSF/Blood glucose ratio 0.26 (0.01-0.53) 0.07 (0.01-0.27) .04

Blood tests

Positive Blood Culture - no. (%) 25/30 (83) 104/197 (53) <.0001

ESR – mm/hr 19 (5-69) 32 (15-58) .24

C-reactive protein – mg/L 193 (137-272) 231 (173-317) .25

Creatinin – μmol/L 228 (95-285) 91 (75-114) <.0001

Sodium – mmol/L 138 (136-140 137 (135-139) .10

Thrombocyte count – 109/L 120 (93-149) 173 (139-225) .004

Data are number/number assessed (%) or median (IQR) unless otherwise stated. CSF white cell count was determined in 242 episodes. CSF protein level was determined in 238 episodes. CSF/blood glucose level was determined in 230 episodes. ESR was determined in 200 episodes. C-reactive protein was determined in 150 episodes. Creatinin was determined in 251 episodes. Sodium was determined in 255 episodes. Thrombocyte count was determined in 245 episodes.

Discussion

The current study provides a detailed description of meningococcal meningitis

from a large prospective cohort study in the Netherlands, aiming to correlate

bacterial genotype, clinical features, prognostic factors, and outcome. Our

findings indicate that meningococcal meningitis remains a serious and life-

threatening disease. The rate of unfavorable outcome of meningococcal

24407 Heckenberg.indd 25 25-02-13 11:11

Chapter 2

26

disease remains substantial at 12%, including a mortality rate of 7%. Neurologic

sequelae in survivors are common (12%), most frequently hearing loss (8%).

A broad spectrum of meningococcal disease was observed in our patients,

ranging from sepsis to meningitis. Signs of systemic disease as indicated by rash,

hypotension, tachycardia, and positive blood cultures occurred frequently in

our patients, frequently resulted in the necessity for cardiopulmonary support

in an intensive care unit, and were associated with unfavorable outcome.

Patients on the meningitis side of the spectrum had a better outcome compared

with those on the sepsis side. In a categorization of the cause of death, sepsis

was the leading cause of death, emphasizing the need for prompt treatment

of systemic complications and the development of new adjunctive therapies

against the septic component of this disease.

Classic symptoms and signs of bacterial meningitis such as headache, fever, neck

stiffness and decreased level of consciousness were absent in many patients.

The classic triad as described in textbooks of fever, neck stiffness, and change

in mental status was not found in two-thirds of patients. This is important

information for physicians who are involved in the identification and treatment

of patients with meningococcal meningitis, and is in line with previous research.11

Initial CSF examination was suspect for bacterial meningitis in most patients.10

Nevertheless, low CSF white cell counts (<100/mm3) were present in ~10% and

were associated with signs of sepsis and unfavorable outcome. In 5 patients,

initial CSF examination of leukocytes, protein, and glucose was entirely normal.

However, meningococcal disease could be identified in all 5 patients through the

presence of rash or the presence of bacteria on Gram staining.

Intravenous antibiotics were started before transportation to the hospital in 2

patients. In the United Kingdom, family doctors are advised to give (parenteral)

antibiotics before transferring the patient to the hospital if meningococcal

meningitis is suspected, but not so in the Netherlands.3, 12 Two problems

arise with treatment before admission to the hospital. First, identification of

patients with meningococcal meningitis by observation of symptoms alone

is difficult.11, 13 Presenting symptoms of meningococcal disease are often

nonspecific, and the current data show that typical signs and symptoms often

do not develop at all.11 Second, it remains unclear whether patients benefit

from such prehospital treatment. Although retrospective data from the United

Kingdom showed a favorable outcome for patients who were treated early

with parenteral antibiotics, prehospital antibiotic treatment of such patients

24407 Heckenberg.indd 26 25-02-13 11:11


27

Ch

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remains controversial.14 A case-control study of children suspected of having

meningococcal disease treated with parenteral antibiotics before admission

showed an association between early treatment and poor outcome.15 While

confounding by severity is a possible explanation for this relation, it has

added to the controversy concerning the strategy of prehospital treatment of

suspected meningococcal disease.16

Cranial CT was a major cause of delay of inhospital administration of therapy.

Antibiotics were not initiated before CT in 83% of patients who underwent

CT before lumbar puncture. We did not specifically record time of delay

of administration of antibiotics, precluding conclusions about a causal

relation between possible delay of treatment and outcome. Nevertheless,

the association between delay in the inhospital administration of antibiotics

and adverse outcome has been shown in previous studies.17, 18 If cranial CT

is to precede lumbar puncture, we recommend that appropriate treatment

(antibiotics with adjunctive dexamethasone) be initiated first.

Dexamethasone was administered in a minority of patients in the current

study. In 2002, a European randomized clinical trial showed that treatment

with adjunctive dexamethasone started before or with the first dose of

antibiotics reduces unfavorable outcome and mortality in adults with bacterial

meningitis.19 This reduction was most obvious in patients with pneumococcal

meningitis. A subsequent meta-analysis including 5 trials involving 623 patients

(pneumococcal meningitis = 234, meningococcal meningitis = 232, other =

127, unknown = 30) showed a reduction of mortality and neurologic sequelae

associated with dexamethasone.20 In meningococcal meningitis, the point

estimate for risk reduction in this meta-analysis was low and not statistically

significant (0.9, confidence interval 0.3–2.1, p=0.7).20 Guidelines differ in their

advice about whether patients with meningococcal meningitis should receive

steroids; some advise the use of steroids in all patients with bacterial meningitis,

others advise discontinuing steroid treatment if the causative pathogen is not

S. pneumoniae.1, 21 Current guidelines by the British Infection Society support

the use of steroids in patients with suspected meningococcal meningitis.12 The

use of high-dose steroids in patients with septic shock may be harmful, and is

therefore not recommended.22

Infection with meningococci belonging to cc11 was associated with sepsis and

poor outcome. We found no additional relations between clinical features and

MLST genotypes. The cc11 was strongly related to the phenotype serogroup

24407 Heckenberg.indd 27 25-02-13 11:11

Chapter 2

28

C and has been associated with elevated levels of disease spreading across

several continents.3, 23 During the study period we noticed an increase in disease

caused by meningococci of cc11 (Figure 1). Since 2002, routine vaccination

with a single dose of conjugated meningococcal C vaccine at 14 months and a

catch-up campaign have reduced the incidence of meningococcal serogroup

C disease in the Netherlands.24

The current study has several limitations. First, only patients with positive

CSF cultures were included. Negative CSF cultures occur in 11%–30% of

patients with bacterial meningitis. Patients in severe septic shock may not

undergo lumbar puncture, as meningococcal sepsis is frequently associated

with coagulation disorders such as disseminated intravascular coagulation.3

In those patients lumbar puncture may not be performed.1 Therefore, these

patients are probably only partly represented in our cohort, probably causing

an underestimation of the rate of sepsis and unfavorable outcome among

our population. Second, a substantial proportion of identified patients with

bacterial meningitis (32%) were not included in our Dutch Meningitis Cohort,

which may also have resulted in selection bias.2

In conclusion, meningococcal meningitis is still a serious and life-threatening

disease. Neuroimaging before lumbar puncture is an important cause of delay

in the administration of antibiotics. Infection with meningococci of cc11 is

related to factors associated with sepsis and to unfavorable outcome.

24407 Heckenberg.indd 28 25-02-13 11:11


29

Ch

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References

1. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354(1):44-53.

2. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351(18):1849-1859.

3. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369(9580):2196-2210.

4. Snape MD, Pollard AJ. Meningococcal polysaccharide-protein conjugate vaccines. Lancet Infect Dis 2005;5(1):21-30.


6. Dodge PR, Davis H, Feigin RD et al. Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis. N Engl J Med 1984;311(14):869-874.

7. van de Beek D, de Gans J. Dexamethasone and pneumococcal meningitis. Ann Intern Med 2004;141(4):327.

8. McMillan DA, Lin CY, Aronin SI, Quagliarello VJ. Community-acquired bacterial meningitis in adults: categorization of causes and timing of death. Clin Infect Dis 2001;33(7):969-975.

9. Neisseria Multi Locus Sequence Typing website. Available at http://pubmlst.org/neisseria/ . Accessed 3 October 2007.

10. Spanos A, Harrell FE, Jr., Durack DT. Differential diagnosis of acute meningitis. An analysis of the predictive value of initial observations. JAMA 1989;262(19):2700-2707.

11. Thompson MJ, Ninis N, Perera R et al. Clinical recognition of meningococcal disease in children and adolescents. Lancet 2006;367(9508):397-403.

12. Heyderman RS. Early management of suspected bacterial meningitis and meningococcal septicaemia in immunocompetent adults--second edition. J Infect 2005;50(5):373-374.

13. Riordan FA, Thomson AP, Sills JA, Hart CA. Who spots the spots? Diagnosis and treatment of early meningococcal disease in children. BMJ 1996;313(7067):1255-1256.

14. Cartwright K, Reilly S, White D, Stuart J. Early treatment with parenteral penicillin in meningococcal disease. BMJ 1992;305(6846):143-147.

15. Harnden A, Ninis N, Thompson M et al. Parenteral penicillin for children with meningococcal disease before hospital admission: case-control study. BMJ 2006;332(7553):1295-1298.

16. Hahne SJ, Charlett A, Purcell B et al. Effectiveness of antibiotics given before admission in reducing mortality from meningococcal disease: systematic review. BMJ 2006;332(7553):1299-1303.

17. Aronin SI, Peduzzi P, Quagliarello VJ. Community-acquired bacterial meningitis: risk stratification for adverse clinical outcome and effect of antibiotic timing. Ann Intern Med 1998;129(11):862-869.

18. Proulx N, Frechette D, Toye B, Chan J, Kravcik S. Delays in the administration of antibiotics are associated with mortality from adult acute bacterial meningitis. QJM 2005;98(4):291-298.


20. van de Beek D, de Gans J, McIntyre P, Prasad K. Steroids in adults with acute bacterial meningitis: a systematic review. Lancet Infect Dis 2004;4(3):139-143.

21. Tunkel AR, Hartman BJ, Kaplan SL et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39(9):1267-1284.

22. Cooper MS, Stewart PM. Corticosteroid insufficiency in acutely ill patients. N Engl J Med 2003;348(8):727-734.


24. de Greeff SC, de Melker HE, Spanjaard L, Schouls LM, van DA. Protection from routine vaccination at the age of 14 months with meningococcal serogroup C conjugate vaccine in the Netherlands. Pediatr Infect Dis J 2006;25(1):79-80.

24407 Heckenberg.indd 29 25-02-13 11:11

24407 Heckenberg.indd 30 25-02-13 11:11

Chapter 3

Naturally occurring lipid A mutants in Neisseria meningitidis

from patients with invasive meningococcal disease are

associated with reduced coagulopathy

Floris Fransen


Hendrik Jan Hamstra

Moniek Feller

Claire J.P. Boog

Jos P.M. van Putten


Arie van der Ende*

Peter van der Ley*

*Both authors contributed equally

PLoS Pathogens, 2009;5(4):e1000396

24407 Heckenberg.indd 31 26-02-13 09:17

Chapter 3

32

Abstract

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis

worldwide. Lipopolysaccharide (LPS), a major component of the Gram-

negative bacterial outer membrane, is sensed by mammalian cells through

Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine

pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the

chemical composition of the lipid A determines how well it is recognized by

TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains,

the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is

generally seen as the prototypical endotoxin-mediated disease. In the present

study, we screened meningococcal disease isolates from 464 patients for their

ability to induce cytokine production in vitro. We found that around 9% of them

were dramatically less potent than wildtype strains. Analysis of the lipid A of

several of the low-activity strains by mass spectrometry revealed they were

penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required

for addition of secondary acyl chains. Sequencing of these genes showed

that all the low activity strains had mutations that inactivated the lpxL1 gene.

In order to see whether lpxL1 mutants might give a different clinical picture,

we investigated the clinical correlate of these mutations in a prospective

nationwide observational cohort study of adults with meningococcal

meningitis. Patients infected with an lpxL1 mutant presented significantly

less frequently with rash and had higher thrombocyte counts, consistent

with reduced cytokine induction and less activation of tissue-factor mediated

coagulopathy. In conclusion, here we report for the first time that a surprisingly

large fraction of meningococcal clinical isolates has LPS with underacylated

lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may

have an important role in virulence by aiding the bacteria to evade the innate

immune system. Our results provide the first example of a specific mutation in

N. meningitidis that can be correlated with the clinical course of meningococcal

disease.

24407 Heckenberg.indd 32 25-02-13 11:11

Lipid A mutAnts in meningococcAL diseAse

33

Ch

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Introduction

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis

worldwide.1 While it is a frequent commensal of the human upper respiratory

tract, in some individuals the bacterium spreads to the bloodstream

causing meningitis and/or sepsis, serious conditions with high morbidity

and mortality. As in all Gram-negative bacteria, lipopolysaccharide (LPS) is

a major component of the outer membrane of N. meningitidis. It is now well

established that LPS is sensed by mammalian cells through Toll-like receptor

4 (TLR4), in combination with coreceptors MD-2 and CD14.2 Activation of this

complex leads to recruitment of the adapters MyD88, Mal, TRIF, and TRAM to

the cytoplasmic domain of TLR4.3 These adapters initiate signal transduction

pathways that lead to induction of innate immunity. These pathways are

classified in a so called “MyD88-dependent” pathway involving MyD88 and

Mal, and a “MyD88-independent” pathway involving TRIF and TRAM. Hallmarks

of MyD88-dependent and MyD88-independent signaling are induction of pro-

inflammatory cytokines and type I IFN respectively. While the response to LPS

can be beneficial to the host by containing a beginning infection, it can also

be detrimental when excessive stimulation occurs through growth of large

numbers of bacteria in the bloodstream as happens during sepsis.2, 4, 5

TLR4 recognizes the lipid A moiety of the LPS molecule.2 The chemical

composition of the lipid A determines how well it is recognized by TLR4 and

consequently it determines the biological activity of the LPS. N. meningitidis

has been reported to produce lipid A with six acyl chains, the optimal number

for TLR4 recognition.6 Indeed purified LPS of this bacterium is highly active and

plasma concentrations of LPS in patients with meningococcal disease correlate

strongly with mortality risk.7 LPS is also important in the activation of the

coagulation system through upregulation of tissue factor. Excessive activation

of the coagulation system can lead to disseminated intravascular coagulation

(DIC), the most feared complication of invasive meningococcal disease.1 DIC

is clinically characterized by hypotension, petechial rash, and depletion of

thrombocytes and coagulation factors.

Uniquely among Gram-negative bacteria, N. meningitidis can grow without

LPS, as was shown by us when we constructed a mutant with an inactivated

lpxA gene, required for the first step in LPS biosynthesis.8 In addition, we have

previously shown that insertional inactivation of the lpxL1 or lpxL2 genes

24407 Heckenberg.indd 33 25-02-13 11:11

Chapter 3

34

required for addition of secondary acyl chains leads to reduced biological

activity of meningococcal LPS.9, 10 The possibility that such mutations might

also occur naturally was suggested to us by a report showing that the group

Y strain HF13 was defective in signaling through the MyD88-independent

pathway and TLR4.11

Here we report that strain HF13 has penta-acylated lipid A due to a mutation

in its lpxL1 gene. Screening of a selection of clinical isolates revealed lpxL1

mutations in approximately 13% of meningococcal disease isolates of all

major serogroups and clonal complexes. Several different kinds of mutations

were found. We also found evidence for on-and-off switching of lpxL1 in vivo

in humans. Importantly, patients with meningococcal meningitis that were

infected with an lpxL1 mutant strain had less severe systemic inflammation and

reduced coagulopathy.

Materials and Methods

Ethics statement

This observational study with anonymous patient data was carried out in

accordance with the Dutch privacy legislation. Written informed consent to use

data made anonymous was obtained from the patient (if possible) or from the

patient’s legal representative.

N. meningitidis strains

Strain HF13 was a kind gift from M. Kilian. The constructed lpxA and lpxL1

mutants were generated in the H44/76 strain as previously described.8, 9 All

other strains were selected from the collection of the Netherlands Reference

Laboratory for Bacterial Meningitis. Details about year of isolation, serogroup,

genotype and anatomical site of isolation are presented in supplementary table

3.1. Meningococci were cultured in GC broth or on GC plates (Difco laboratories)

supplemented with 1% (vol/vol) Vitox (Oxoid) at 37ºC in humified atmosphere

of 5% CO2.12 Bacteria were suspended in PBS and the A

620 was determined.

The bacteria were heat inactivated at 56ºC for 30 min. Serogrouping were

performed as described elsewhere.13 MLST was performed as described by

Maiden et al.14

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Lipid A structure

Bacteria were grown as described above and suspended in isobutyric acid-

ammonium hydroxide 1M (5:3, v/v). Lipid A was extracted as described

previously with slight modifications.15 The lipid A structure was analyzed by

nanoelectrospray tandem mass spectrometry (MS/MS) on a Finnigan LCQ in

the negative (MS) or positive (MS/MS) ion mode.16

Sequencing

DNA was extracted from boiled cultures of N. meningitidis. Sequencing of

lpxL1 was carried out using primers 344-2 and 670-1 (supplementary table

3.2) and BigDyeTerminator chemistry (Applied Biosystems) according to

the instructions of the manufacturer. The primers used to obtain sequences

upstream and downstream of lpxL1 are presented in supplementary table 3.2.

Sequence traces were obtained with ABI Big-dyes and an ABI 3730 sequencer.

Cell lines and PBMCs

PBMC from HLA-oligotyped donors after leukapheresis were isolated by

centrifugation of buffy coat cells on Ficoll-Hypaque (Pfizer) and were used

after cryopreservation. For experiments and/or maintenance, the human

monocyte cell line Mono-mac-6 (MM6), the mouse macrophage cell line

J774A.1, and PBMCs were suspended in IMDM (Gibco BRL) supplemented

with 100 units/ml penicillin, 100 μg/ml streptomycin, 300 μg/ml l-glutamine

(Gibco BRL), and 10% heat-inactivated fetal calf serum (FCS) (Gibco BRL).

For experiments and maintenance of HEK-293 cells stably transfected with

human TLR4A, MD-2, and CD14 (Invivogen), DMEM (Gibco BRL) was used,

supplemented with 10% FCS, 10 µg/ml blasticidin (Invivogen), and 50 µg/ml

Hygromycin B (Invivogen).

ELISA

Depending on the experiment either J774A.1, MM6, PBMCs, or HEK-293 hTLR4/

MD-2/CD14 cells were used. Different plates and quantities of cells were used:

1.106 cells in 1 ml medium per well in 12-well plates, 9.104-5.105 cells in 250-

1000 µl medium per well in 24-well plates, and 1.105-3.105 cells in 200-300 µl

medium per well in 96-well plates. Cells were stimulated with bacteria and

incubated o/n at 37 °C in a humidified atmosphere containing 5% CO2. Cytokine

concentrations in the culture supernatants were quantified with ELISA. Mouse

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IP-10 was determined with mouse IP-10 ELISA kit (R&D systems) and human IL-

6, TNF-α, IL-1β, and IL-8 with PeliPairTM reagent sets (Sanquin).

Meningitis cohort study

The Dutch Meningitis Cohort Study included 258 patients with meningococcal

meningitis; from 254 patients the bacterial strain was stored in the Netherlands

Reference Laboratory for Bacterial Meningitis.17 Inclusion and exclusion criteria

have been described extensively elsewhere.13 In summary, eligible patients

were older than 16 years, had bacterial meningitis confirmed by culture of

cerebrospinal fluid (CSF), and were listed in the database of the Netherlands

Reference Laboratory for Bacterial Meningitis from October 1998 to April

2002. This laboratory receives CSF isolates from about 85% of all patients with

bacterial meningitis in the Netherlands. The treating physician was contacted,

and informed consent was obtained from all participating patients or their

legally authorized representatives. This observational study with anonymous

patient data was carried out in accordance with the Dutch privacy legislation.

Patients underwent a neurologic examination at discharge, and outcome

was graded with the Glasgow Outcome Scale. This measurement scale is well

validated with scores varying from 1 (indicating death) to 5 (good recovery). A

favorable outcome was defined as a score of 5, and an unfavorable outcome as

a score of 1-4. Focal neurologic deficits were defined as focal cerebral deficits

(aphasia, monoparesis, or hemiparesis) or cranial nerve palsies. Serogrouping,

MLST, and susceptibility testing of meningococcal isolates were performed by

the Netherlands Reference Laboratory for Bacterial Meningitis.

Statistics

The Mann-Whitney U test was used to identify differences between groups in

continuous variables, and dichotomous variables were compared by the chi-

square or Fisher exact test. All statistical tests were 2-tailed, and a p value less

than 0.05 was regarded as significant.

List of accession numbers/ID numbers for genes mentioned in the text

Please see supplementary table 3.3 for accession numbers.

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Results

Strain HF13 is a natural lpxL1 mutant

Mogensen et al. demonstrated that the serogroup Y strain HF13 is defective

in TLR4 activation and initiation of MyD88-independent signaling.11 Reduced

biological activity of meningococcal LPS is associated with altered lipid A

structure.9, 10 Therefore, the lipid A structure of strain HF13 was assessed by mass

spectrometry (Figure 1A). The spectrum shows major peaks that correspond

with lipid A with only five acyl chains. One of the two secondary C12

acyl chains

is absent, but the spectrum is not conclusive on which one, since the C12

acyl

chains have the same mass. This result implies that in strain HF13 either lpxL1

or lpxL2 is inactive, as we previously found that the addition of the secondary

C12

acyl chains to lipid A requires active lpxL1 and lpxL2.9 Sequence analyses of

both genes showed a normal lpxL2 sequence, but the lpxL1 sequence contained

one adenosine deletion in a poly adenosine tract, leading to a frameshift and a

premature stop of the translated protein (Figure 2, Table 1).

The inactivated lpxL1 gene in strain HF13 results in a penta-acylated lipid

A lacking the secondary acyl chain at the 2’-position in lipid A, while N.

meningitidis typically has a hexa-acylated lipid A (Figure 1B). These results

provide an explanation for the inability of strain HF13 to activate TLR4 and to

initiate MyD88-independent signaling.

Mutations in lpxL1 are present in several serogroups and clonal complexes

To evaluate the distribution of lpxL1 mutations among meningococcal isolates

from patients, we initially screened a panel of 56 serogroup Y meningococcal

isolates for their capacity to induce the MyD88-independent cytokine IP-10

in the mouse macrophage cell line J774A.1 (supplementary figure 3.1). As

controls, strain H44/76 and HF13 were included. Of 56 serogroup Y isolates,

eight strains induced like HF13 little or no IP-10. Sequence analyses of lpxL1

of these isolates revealed that they all had mutations in lpxL1, resulting in an

inactive gene. Five strains had one adenosine deletion in a poly A tract just

like strain HF13 (type V mutation, Figure 2, Table 1), two strains had a deletion

of ten nucleotides (type VI mutation), and one strain had an insertion of the

insertion element IS1301 (Type I mutation).

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Figure 1. Strain HF13 is an lpxL1 mutant

(A) Mass spectrum of HF13 lipid A. The highest peak (1653.6) corresponds to penta-acylated lipid A with two phosphate groups and one phosphoethanolamine (PEA), the second peak (1530.4) corresponds to penta-acytlated lipid A with two phosphate groups without PEA. (B) Depiction of N. meningitidis wildtype lipid A. The acyl chain that is added by LpxL1 is indicated.

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Figure 2. Presentation of all lpxL1 mutations

The lpxL1 gene sequence of strain MC58 is shown including the different types of mutations and their position in the gene found among isolates from patients. Each type of mutation is indicated with a different color. An ‘_’ indicates a nucleotide that is deleted in the mutant strain, pink indicates an insertion element (type I and II mutations), yellow indicates a deletion of a guanine in a stretch of five guanines (type III mutation), green indicates a deletion of an adenosine in a stretch of five adenosines (type IV mutation), light blue indicates a deletion of an adenosine in a stretch of seven adenosines (type V mutation), dark blue indicates an insertion of an adenosine in a stretch of seven adenosines (type V mutation) and the sequences that are highlighted in red or gray are deleted in the mutant strain (type VI and VII mutations respectively).

Table 1. List of all lpxL1 mutant strains

Strain no. Isolated from serogroup ST Clonal complex Type of mutation in lpxL1a

lpxL1 mutants among 56 serogroup Y isolates

HF13 Ndb Y nd nd V

2011169 blood Y 23 23 V

2040760 blood Y 23 23 V

970455 blood Y nd nd V

2050913 joint puncture Y 2786 23 V

971523 CSF Y nd nd V

971886 CSF Y nd nd VI

982195 blood Y nd nd VI

2040608 CSF Y nd nd I

lpxL1 mutants among 114 isolates representing major serogroups and clonal complexes

2000569 blood X 750 750 V

2011833 blood C 3553 269 V

2041268 blood B 4926 35 V

2050093 blood B 461 461 V

2030162 blood C 337 41/44 V

2051372 blood B 461 461 V

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Strain no. Isolated from serogroup ST Clonal complex Type of mutation in lpxL1a

2010151 blood C 461 461 IV

2021270 CSF C 461 461 III

2041396 CSF B 4930 18 III

2050806 CSF B 213 213 III

2071416 blood Y 23 23 VI

2020799c CSF B 35 35 conserved amino acid change

2050392 CSF B 213 213 VII

lpxL1 mutants among multiple isolates from a single patient

Patient 94176

941761 I CSF C nd nd conserved amino acid change

941761 III Troat swab C nd nd wildtype

Patient 9707010

970710 I CSF C nd nd wildtype

970710 III nose swab C nd nd IV

Patient 971859

971859 I CSF C nd nd IV

971859 III Throat swab C nd nd wildtype

lpxL1 mutants among isolates from 254 patients in the prospective cohort study

2012202 CSF B 41 41/44 V

2020434 CSF C 11 11 V

991174 CSF C 11 11 V

990576 CSF B 571 41/44 V

991382 CSF B 191 41/44 V

2011833 CSF C 3553 269 V

991344 CSF B 42 41/44 III

2000607 CSF B 40 41/44 III

2000311 CSF B 461 461 III

991093 CSF B 5451 32 III

2020622 CSF B 5458 41/44 IV

990344 CSF B 5449 41/44 IV

2010640 CSF B 1474 41/44 conserved amino acid change2011334 CSF C 11 11 II

2011764 CSF B 303 41/44 conserved amino acid change

992008 CSF B 146 41/44 Not detected

aType of mutations found in lpxL1. Colors in parentheses correspond to colors shown in figure 2. Type I mutation: insertion of IS1301 (pink), type II mutation: insertion of IS1655 (pink), type III mutation: deletion of a guanine in a stretch of five guanines (yellow), type IV mutation: deletion of an adenosine in a stretch of five adenosines (green), type V mutation: deletion or insertion of an adenosine in a stretch of seven adenosines (light and dark blue), type VI mutation: deletion of ten nucleotides (red), type VII mutation: deletion of C-terminal part of the lpxL1 gene (gray). For all strains with conserved amino acid changes, the inactivation of lpxL1 has been confirmed with analysis of the lipid A by mass spectrometry. bNd: not determined. cStrain 2020799 was part of both the panel of 114 isolates representing all major serogroups and clonal complexes and the panel of 254 isolates from patients in the prospective cohort study.

Table 1 (continued)

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These results prompted us to investigate the distribution of lpxL1 mutations

among meningococci of the major serogroups and clonal complexes.

Previously, we have shown that at higher dilutions an lpxL1 mutant induces

less pro-inflammatory cytokines than wildtype N. meningitidis.9, 10 To identify

meningococcal isolates with mutations in lpxL1, isolates were tested on their

capacity to induce IL-6 in the human monocytic cell line Mono Mac 6 (MM6). Of

114 isolates, representing all major serogroups and clonal complexes, 13 were

found to induce low amounts of IL-6 (supplementary figure 3.2). Sequence

analyses of lpxL1 showed that 12 isolates had a mutation in lpxL1, rendering the

gene inactive (Figure 2, Table 1). Of these strains, 10 had an insertion or deletion

in a polyadenosine or polyguanosine tract (type III, IV and V mutations); six of

these had the same mutation as found in the majority of mutant serogroup

Y strains. One strain had a type VI mutation, like in the two aforementioned

serogroup Y strains. One strain had a deletion of the C-terminal part of the gene

(type VII mutation). The remaining strain (2020799) had apparently no mutation

in lpxL1 that would lead to its inactivation. However, closer examination of its

putative amino acid sequence showed that one amino acid was altered at a

position conserved in all known lpxL1 homologues. Therefore, the LpxL1

protein of this strain is probably nonfunctional. Indeed, we confirmed that

strain 2020799 had penta-acylated lipid A by mass spectrometry (data not

shown). As a control, also lpxL1 of 34 strains that induced a normal level of

IL-6 was sequenced. As expected, these strains had no mutations in lpxL1 (data

not shown). Together, seven unique lpxL1 mutations were found among this

panel of different serogroups and different clonal complexes, indicating that

inactivation of lpxL1 must have occurred multiple times independently. The

results show that lpxL1 mutations are not associated with serogroup or clonal

complexes and occur also among the serogroup B and C strains, which are

prevalent among isolates from patients with meningococcal disease in Europe.

Screening of lpxL1 mutations in a panel of multiple isolates per patient

Most of the identified lpxL1 mutations were in nucleotide repeats of adenosines

and guanosines, the type III, IV and V mutations (Figure 2, Table 1). These

sequences are prone to cause slippage of the DNA polymerase during DNA

replication, leading to reversible frameshift mutations. This slipped-strand

mispairing is the most common mechanism of translational phase variation,

the process of random and reversible on-and-off switching of a gene. Phase

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variation creates a phenotypically diverse population, allowing the bacterium

to adapt to different microenvironments within the human host. To investigate

whether N. meningitidis can switch lpxL1 on-and-off we screened a panel of

strains obtained from different anatomical locations within individual patients:

isolates from the blood and/or cerebrospinal fluid (CSF) as well as from the

throat and/or nose of 40 patients were used. The MM6 cell line was stimulated

with these strains and IL-6 production was measured with ELISA. Three strains

induced low levels of IL-6 compared to wildtype N. meningitidis (supplementary

figure 3.3). These isolates were from three different patients. Two strains were

isolated from the cerebrospinal fluid and one strain was isolated from the

throat. The other isolates of these patients induced normal levels of IL-6. The

lpxL1 genes of all isolates of these three patients were sequenced and found

to be mutated in the isolates that induced low IL-6, but not in the isolates that

induced normal IL-6 (Figure 2, Table 1). Two strains had a type IV mutation,

which potentially is reversible. The third strain had a point mutation leading to

substitution of a conserved amino acid. These results suggest that in the host

the expression status of lpxL1 of meningococci is subject to phase variation.

lpxL1 mutants induce less pro-inflammatory cytokines in a TLR4-dependent manner

The identified lpxL1 mutations occurred in strains of widely varying genetic

background, and it is therefore conceivable that other factors besides altered

LPS contribute to their reduced cytokine induction. To investigate this, titrations

of four of the spontaneous lpxL1 mutants were compared in their capacity to

induce cytokines in MM6 cells with titrations of our previously constructed

lpxL1 knockout mutant and its parent strain H44/76, as well as the completely

LPS-deficient strain pLAK33 (Figure 3). Clearly, the LPS-deficient strain pLAK33

is much less potent in inducing IL-6 than the wildtype strain H44/76. IL-6

induction by the constructed lpxL1 mutant is similar to that by pLAK33 and the

four lpxL1 mutants isolated from patients.

To demonstrate that the lpxL1 mutants induced less cytokines than wildtype

strains because their LPS is less well recognized by the LPS receptor complex,

titrations of a similar panel of strains was used to stimulate HEK293 cells

transfected with human TLR4, MD-2, and CD14. Activation of the receptor

complex was assessed by measuring IL-8 production (Figure 4). Wildtype

strain H44/76 was much more efficient in TLR4 activation than the mutants.

All lpxL1 mutants, either constructed or isolated from patients, showed a

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similar decrease in IL-8 induction, while the LPS-deficient pLAK33 cells were

even less active. Together, these results demonstrate that the lpxL1 mutants

activate human TLR4 less efficiently, and this is the sole reason for their reduced

biological activity.

We have shown that lpxL1 mutants induce less cytokines in human and

murine cell lines. However, these in vitro models do not necessarily represent

the situation in vivo and do not take into account the genetic diversity of the

human population. To mimic a systemic meningococcal infection more closely,

also human peripheral blood mononuclear cells (PBMCs) of several donors

were stimulated with titrations of a selection of N. meningitidis strains. After

stimulation, concentrations of IL-6, TNF-α, and IL-1β were determined in the

supernatant (Figure 5). These pro-inflammatory cytokines are known to mediate

the toxic effects of LPS.2 In all donors, wildtype strain H44/76 induced much

more IL-6, TNF-α, and IL-1β than the mutants. Overall, the constructed and

spontaneous lpxL1 mutants showed a similar reduction in cytokine induction.

Meningitis patients infected with lpxL1 mutant meningococci have reduced

inflammation and coagulopathy

We next explored whether infection with lpxL1-mutant meningococcal strains

was associated with a particular clinical phenotype. The meningococcal isolates

from 254 patients from a prospective nationwide observational cohort study of

696 adults with community-acquired bacterial meningitis in the Netherlands

(period, 1998-2002)13, 17 were analyzed for their ability to induce IL-6. Of the

254 isolates, 172 (68%) were of serogroup B, 78 (31%) of serogroup C, 3 (1%) of

serogroup Y, and one (<1%) of serogroup W135. Multilocus sequence typing

showed 91 unique sequence types. The most prevalent clonal complexes were

cc41/44 (41%), cc11 (24%), and cc32 (16%).17

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Figure 3. Comparison of IL-6 induction in MM-6 cells between wildtype strains and lpxL1 mutants

MM-6 cells were stimulated for 18 h with titrations of indicated strains and IL-6 in supernatant was quantified with ELISA. H44/76 is a wildtype strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of three independent experiments are shown. Error bars indicate S.E.M. of triplicates.

Figure 4. Comparison between wildtype strains and lpxL1 mutants of IL-8 induction in HEK293 cells transfected with human TLR4

HEK293 cells transfected with human TLR4, CD14, and MD-2 were stimulated for 18 h with titrations of indicated strains and IL-8 in supernatant was quantified with ELISA. H44/76 is a wildtype strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of three independent experiments are shown. Error bars indicate S.E.M. of triplicates.

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Figure 5. Comparison between wildtype strains and lpxL1 mutants in pro-inflammatory cytokine induction in PBMCs

PBMCs from three different donors were stimulated with titrations of the indicated strains and IL-6, TNF-α, and IL-1β were quantified in the supernatant 18 h after stimulation. H44/76 is a wildtype strain, L8 lpxL1 is a constructed lpxL1 mutant, pLAK33 is an LPS-deficient mutant, and all other strains are spontaneous lpxL1 mutants. Results of one representative experiment of two independent experiments are shown. Error bars indicate S.E.M. of triplicates.

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MM6 cells were stimulated with these strains and IL-6 induction was assessed

(supplementary figure 3.4). The isolates of 17 patients (7%) showed a decreased

IL-6 induction and sequencing revealed mutations in lpxL1 in all but one

(Figure 2, Table 1). Twelve isolates had a type III, IV or V mutation. Three strains

had a point mutation leading to substitution of an essential amino acid, and

one strain had an IS1655 insertion. In one strain (992008) we were unable to

identify a mutation in lpxL1 that could lead to gene inactivation or inactive

gene product. Further analyses with mass spectrometry to determine the mass

of its lipid A and silver staining of a Tricine-SDS-PAGE gel to analyze the size

and quantity of its LPS, demonstrated that LPS was not detectable in this strain

(results not shown). The responsible mutation remains to be identified. There

were no overall differences in lpxL1 mutation frequency between serogroups

(P=0.85) and clonal complexes (P=0.56).

Next, we correlated results of the mutation analysis with clinical data (Table 2).13,

17 Patients infected with lpxL1 mutant strains tended to be younger (P=0.053)

and to present less frequently with fever (P=0.057). None of the patients infected

with an lpxL1 mutant strain presented with hypotension and these patients

had correspondingly lower levels of serum creatinine. They were less likely to

present with rash compared with those infected with wildtype meningococci

(5/16 [31%] vs. 157/236 (67%); P=0.006; Figure 6) and had higher platelet counts

(P=0.005). Rash was strongly related with lower platelet counts (P<0.0001). To

investigate the possibility that the clinical differences found between the two

patient groups were confounded by the different ages of the patient groups,

a multivariate analysis adjusting for age was performed. The difference in

platelet count (P=0.003) and rash (P=0.004) remained statistically significant

after adjusting for age. Subgroup analysis of clonal complex 41/44 showed

similar results. The differences in platelet count (P=0.007), rash (P=0.006), and

age (P=0.053) between patients infected by mutant and wildtype strains were

also present in the subgroup of clonal complex 41/44.

None of the patients infected with lpxL1 mutant strains developed septic shock

during clinical course, while 13% of the wildtype-infected patients did. One

patient infected with an lpxL1 mutant strain died of respiratory failure after

multiple seizures. By contrast, sepsis was the leading cause of death among

patients infected with wildtype meningococci (14 of 16 fatalities, 88%).

Thus, the lpxL1 mutation occurs frequently among meningococci causing

meningitis. Patients infected by mutant strains have a clinical phenotype

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consistent with less systemic inflammation and reduced activation of the

coagulant system.

Table 2. Clinical features of 254 adults with meningococcal meningitis due to lpxL1 mutant and wildtype strains

Characteristic Wildtype (N=237) lpxL1 mutante (N=17) P-valuef

Admission

Age in yr - median (IQRa) 31 (19-51) 21 (19-30) 0.053

Fever (temperature >38.0 ºC) – no. (%) 87/231 (38) 2/16 (13) 0.057

Neck stiffness – no. (%) 205/234 (88) 17/17 (100) 0.23

Median score on GCSb (IQR) 13 (10-15) 12 (11-15) 0.97

Rash 157/236 (67) 5/16 (31) 0.006

Hypotension (Systolic BPc <90mmHg) – no. (%)

23/204 (11) 0/16 (0) 0.39

Focal neurological deficits – no. (%) 51/237 (22) 2/17 (12) 0.54

Laboratory investigations

Cerebrospinal fluid white cell count – median per mm3 (IQR)

5205 (1466-12605)

5376 (3063-11416)

0.89

Positive blood culture – no. (%) 117/207 (49) 11/16 (69) 0.44

Platelet count – median 109/L (IQR) 162 (123-211) 215 (169-270) 0.005

Serum creatinine – median µmol/L (IQR) 95 (77-128) 84 (69-99) 0.051

Clinical course

Septic shock – no. (%)d 30/237 (13) 0/17 (0) 0.23

Neurologic complication – no. (%) 98/237 (41) 5/17 (29) 0.45

Outcome

Death – no. (%) 18/237 (8) 1/17 (6) 1.00

Focal neurological deficits – no. (%) 25/218 (11) 3/16 (19) 0.42

Unfavorable outcome – no. (%) 27/237 (11) 3/17 (18) 0.43aIQR denotes interquartile range, bGCS Glasgow Coma Scale, cBP blood pressure. dSeptic shock was defined as systolic blood pressure <90 mmHg with positive blood culture. Systolic blood pressure was measured on admission in 243 patients, GCS in 253 patients, CSF white cell count in 238 patients, and serum platelet count in 241 patients. eIn 16 strains a mutation in lpxL1 was found, but not in strain 992008. fThe Mann-Whitney U test was used to identify differences between groups in continuous variables, and dichotomous variables were compared by the chi-square or Fisher exact test.

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Figure 6. Clinical correlate of lpxL1 mutations in meningococcal meningitis

(A) Frequency of rash in patients presenting with meningitis infected by lpxL1 wildtype and mutant strains. (B,C) Platelet counts on admission for lpxL1 wildtype and mutant strains (B) and patients presenting with and without rash (C). Horizontal bars reflect medians. The Mann-Whitney U test was used to identify differences between groups in continuous variables, and dichotomous variables were compared by the chi-square or Fisher exact test.

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Discussion

Overall, we screened meningococcal isolates of 464 different patients and

identified 40 strains with an lpxL1 mutation. An additional low-activity strain had

no lpxL1 mutation but appeared to be LPS-deficient; the responsible mutation

is currently under investigation. Thus, 8.6% of patients were infected with an

lpxL1 mutant, which is surprisingly common. There are several lines of evidence

making it very likely that lpxL1 mutants arise spontaneously in the host instead

of introduction of an lpxL1 mutation into one or several clones and subsequent

spreading among the meningococcal population. Firstly, lpxL1 mutations exist

in isolates of many serogroups and clonal complexes. Secondly, we identified

12 unique mutations in lpxL1. Thirdly, most lpxL1 mutations (71%) are due to

frameshifts in homopolymeric nucleotide tracts, making phase variation likely.

Finally, we found evidence for switching from wildtype to lpxL1 mutant in vivo in

patients for which multiple isolates were available.

A picture emerges of N. meningitidis modulating its lipid A structure under

selective pressure. Under some conditions, hexa-acyl lipid A has to be beneficial

to compensate for enhanced recognition by the innate immune system. Lipid

A with six acyl chains can protect bacteria from the antibacterial molecules in

mucosal secretions, consistent with the observation that many bacteria inhabiting

the respiratory tract and gut still produce hexa-acyl LPS.18 Chronic inflammation

of these environments due to LPS stimulation is probably prevented because

epithelial cells express low levels of either TLR4, MD-2, or CD14 at the mucosal

surface. On the other hand, the submucosal spaces are normally sterile and the

defense cells present there, such as macrophages, dendritic cells, and neutrophils,

express all the components of the LPS receptor complex and can therefore

respond potently after an encounter with a Gram-negative bacterium.18 Perhaps

for this reason most species of Gram-negative bacteria with hexa-acyl lipid A that

inhabit the mucosal surfaces rarely become invasive. On the other hand, many

Gram-negative pathogens that cause systemic infection do not produce hexa-

acyl lipid A. Most of these bacteria have other habitats than the mucosa and

enter the body via nonmucosal routes.6 A good example is the plague bacillus

Yersinia pestis. At mammalian body temperature Y. pestis normally produces

tetra-acyl LPS that is poorly recognized by TLR4. Interestingly, a modified strain

that produced hexa-acyl LPS at 37 ºC was no longer virulent in wildtype mice but

fully virulent in TLR4-deficient mice, demonstrating the importance of evasion

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of TLR4 activation for this bacterium.19 N. meningitidis seems to be one of the

exceptions to the general rule that Gram-negative bacteria with hexa-acyl lipid

A do not cause systemic disease. However, our observation that a proportion

of clinical isolates have penta-acylated LPS suggests that evasion of TLR4

activation might aid the bacterium to circumvent host defences after crossing

the nasopharyngeal epithelium. The hypothesis that TLR4 plays an important

role in the prevention of meningococcal disease corroborates with the finding

that subjects with rare TLR4 mutations have an increased risk for developing the

disease.20 If the assumption is correct that hexa-acyl LPS gives the bacterium

an advantage on mucosal surfaces and that non hexa-acyl LPS is better for

bacteria in submucosal spaces, one would expect that the frequency of lpxL1

mutants is lower in meningococcal isolates from the respiratory tract compared

to meningococcal isolates from the cerebrospinal fluid or blood.

Mogensen et al. showed that strain HF13 is specifically defective in activation of

the MyD88-independent pathway, but not in inducing the MyD88-dependent

pathway.11 However, we demonstrate that strain HF13 and other lpxL1 mutants

are also defective in inducing the MyD88-dependent cytokines IL-6, TNF-α,

and IL-1β. Our experiments indicate that lpxL1 mutants or purified lpxL1 LPS

compared to wildtype controls are not specifically deficient in inducing the

MyD88-dependent vs. independent pathway. This apparent discrepancy might

be explained by the dose of bacteria used. If cells are stimulated with a high dose

of bacteria the difference between lpxL1 mutant and wildtype is only detectable

for the MyD88-independent pathway. This is because LPS is the only bacterial

component capable of inducing the MyD88-independent pathway, while

many other bacterial components can induce the MyD88-dependent pathway

(e.g. TLR2 ligands). When cells are stimulated with lower doses of bacteria the

difference in induction of the MyD88-dependent pathway becomes apparent,

because LPS is by far the most active component of the bacterium and the other

non-TLR4 ligands that can activate the MyD88-dependent pathway are diluted

too far to be still active.

The relatively high frequency of phase variation raises the question whether

the lpxL1 mutations might have arisen in vitro after isolation from the patient.

Previously, we have performed extensive research on the phase variation of

porA in N. meningitidis. In this gene, homopolymeric nucleotide tracts are found

in the promoter (polyguanidine) and in the coding region (polyadenine). The

frequencies by which these sequences vary in length are 10-3.12, 21 Others showed

24407 Heckenberg.indd 50 25-02-13 11:11


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phase variation of capsule expression caused by insertion of IS1301 in the siaA

gene with a frequency of phase variation of 9×10−4.22, 23 In vitro selection of porA

phase variants and siaA phase variants have not been reported. Meningococcal

isolates received by the Netherlands Reference Laboratory for Bacterial Meningitis

(NRLBM) are low passages (up to 2 passages). We sequenced the lpxL1 gene of 20

individual colonies of a culture of a mutant isolate (971859 I) and of 25 individual

colonies of a culture of isolate 971859 III and found in each instance the same

sequence, i.e. 20 mutant sequences and 25 wildtype sequences, respectively.

Therefore, we estimate the frequency of phase switching to be less than 2.2 x

10-2. In addition, we sequenced lpxL1 of DNA extracted from a swap taken from

4 different quadrants of another culture plate of isolate 971859 III. All 4 lpxL1

sequences were homogeneous and identical. Thus we are confident that the

discovered lpxL1 mutations are not caused by in vitro phase variation.

Infection with lpxL1-mutant meningococcal strains is associated with a particular

clinical phenotype, which consisted of less systemic inflammation and reduced

activation of the coagulant system, reflected in less fever, higher serum platelet

counts, and lower numbers with rash. Moreover, our in vitro data have shown

that lpxL1 mutants induce much less pro-inflammatory cytokines than wildtype

strains. The coagulation system is activated through upregulation of tissue factor.1

It has been demonstrated that LPS upregulates tissue factor on monocytes and

endothelial cells.24-26 Furthermore, in particular the pro-inflammatory cytokine

IL-6 appears to mediate in vivo expression of tissue factor. 27, 28 Finally, IL-1β and

TNF-α inhibit anticoagulant pathways by downregulating thrombomodulin at

the endothelial surface and by increasing plasminogen activator inhibitor type-1

(PAI-1).29, 30 Thus, our finding that patients infected with an lpxL1 mutant show

less activation of the coagulation system is consistent with our results that show

that lpxL1 LPS is less potent and that lpxL1 mutants induce less pro-inflammatory

cytokines. Remarkably, the lpxL1 mutants induced the same degree of CSF

leukocytosis as wildtype strains. There are several explanations for “normal” CSF

white cell counts in patients infected by mutant strains. Patients in the cohort

all had positive CSF cultures; almost all had clinical signs of meningitis and CSF

leukocytosis. Likely, leukocytosis is not only mediated by lipid A, but also by other

microbial constituents.

It should be noted that not all groups of patients were included in our analysis

of clinical patient data. The study only included adults with meningitis. Patients

younger than 16 years or patients with sepsis only were not included. Therefore,

24407 Heckenberg.indd 51 25-02-13 11:11

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52

our results are potentially biased by excluding these patient groups. Patients

with meningitis often have a less severe form of the disease, as reflected by the

overall low mortality of 8% in our study. However, patients with sepsis have

very serious symptoms resulting from high concentrations of bacteria in the

circulation. Mortality rates in these patients can be as high as 50%. Also, patients

younger than 16 years are an import group, because rates for meningococcal

disease are highest for young children.1 It would be interesting to see whether

lpxL1 mutants also exist in these patients groups, and if so, if these patients

have a different clinical course compared to patients infected with a wildtype

strain. These additional data are needed to fully understand the impact of lpxL1

mutations on meningococcal disease.

Meningococcal sepsis is generally seen as the prototypical endotoxin-mediated

disease. Here we report for the first time that meningococcal lipid A mutants

which are defective in TLR4 activation occur naturally. Their frequency is

unexpectedly high, suggesting an important role in virulence for the resulting

low-activity LPS. Our results suggest that in most cases this mutation has occurred

through phase variation, and may give the bacteria an advantage because

they are less well sensed by the innate immune system. Patients infected with

these mutant strains endure milder symptoms with less systemic inflammation

and reduced activation of the coagulant system, showing that our findings are

clinically relevant. Importantly, these results with lpxL1 also provide the first

example of a specific bacterial mutation which can be associated with the clinical

course of meningococcal disease. More generally, it shows how there can be an

underestimated heterogeneity in the TLR4-activating capacity of pathogenic

bacteria.

Acknowledgements

M. Kilian kindly provided N. meningitidis group Y strain HF13. This publication

made use of the Neisseria Multi Locus Sequence Typing website (http://pubmlst.

org/neisseria/) developed by Keith Jolley and Man-Suen Chan and sited at the

University of Oxford 31. The development of this site has been funded by the

Wellcome Trust and European Union. We thank M. Bertayli, H.D. Meiring, and J.

ten Hove for experimental assistance.

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Supplementary material

Supplementary figure 3.1. Screening of N. meningitidis group Y clinical isolates on cytokine induction

J774A.1 cells were stimulated for 3 h with a panel of group Y strains (0.1 OD) and IP-10 in the supernatant was determined with ELISA.

Supplementary figure 3.2. Screening of panel of N. meningitidis clinical isolates representing all major serogroups and clonal complexes

24407 Heckenberg.indd 53 26-02-13 09:17

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54

MM6 cells were stimulated for 18 h with a selection of clinical isolates (0.001 OD) representing all serogroups and clonal complexes. IL-6 was determined in the supernatant with ELISA.

Supplementary figure 3.3. Screening of panel of multiple isolates per patient

MM6 cells were stimulated for 18 h with a panel of clinical isolates (0.001 OD), of which multiple isolates were obtained from a single patient. IL-6 was determined with ELISA.

supplementary figure 3.2 (continued)

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Supplementary figure 3.4. Screening of clinical isolates of patients included in the Dutch meningitis cohort study

MM6 cells were stimulated for 18 h with 254 isolates from patients with meningitis (0.001 OD). IL-6 was determined with ELISA.

24407 Heckenberg.indd 55 25-02-13 11:11

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Supplementary table 3.1. The list of meningococcal strains used in this study is accessible at http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000396#s5

Supplementary table 3.2. List of primers used for the sequencing of lpxL1

Primer Sequence coordinates(according to MC58 sequence)a

AvdE_02NMB1417F 1452953 - 1452971

344-2 1453690 - 1453709

AvdE_NMB1419R 1454685 - 1454669

670-1 1454693 - 1454674

AVDE-LPX1-101 1454977 - 1454960

AVDE_LPX1_100 1455059 - 1455039aaccession number AE002098.2.

Supplementary table 3.3 List of accession numbers/ID numbers for genes mentioned in the text

Strain Number Accession number

992073 FJ472279

9718866 FJ472280

9821956 FJ472281

2040760 FJ472282

2011169 FJ472283

2010151 FJ472284

2000569 FJ472285

2021270 FJ472286

2030162 FJ472287

2041268 FJ472288

2041396 FJ472289

2050093 FJ472290

2050806 FJ472291

2051372 FJ472292

2071416 FJ472293

2050392 FJ472294

970455 FJ472295

2050913 FJ472296

971523 FJ472297

2000311 FJ472298

2000607 FJ472299

2010640 FJ472300

2011764 FJ472301

Strain Number Accession number

2011833 FJ472302

2012202 FJ472303

2020434 FJ472304

2020622 FJ472305

2020799 FJ472306

990344 FJ472307

990576 FJ472308

991093 FJ472309

991174 FJ472310

991344 FJ472311

991382 FJ472312

992008 FJ472313

971859_I FJ472314

971859_III FJ472315

970710_I FJ472316

970710_III FJ472317

941761_I FJ472318

941761_III FJ472319

2040608_5# FJ472320

2040608_3# FJ472321

2011334_5# FJ472322

2011334_3# FJ472323

24407 Heckenberg.indd 56 25-02-13 11:11


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References


2. Beutler B, Rietschel ET. Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol 2003;3(2):169-176.

3. Palsson-McDermott EM, O’Neill LA. Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 2004;113(2):153-162.

4. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 1993;328(20):1471-1477.

5. Russell JA. Management of sepsis. N Engl J Med 2006;355(16):1699-1713.

6. Munford RS, Varley AW. Shield as signal: lipopolysaccharides and the evolution of immunity to gram-negative bacteria. PLoS Pathog 2006;2(6):e67.

7. Brandtzaeg P, Bjerre A, Ovstebo R, Brusletto B, Joo GB, Kierulf P. Neisseria meningitidis lipopolysaccharides in human pathology. J Endotoxin Res 2001;7(6):401-420.

8. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, van der Ley P. Meningitis bacterium is viable without endotoxin. Nature 1998;392(6675):449-450.

9. van der Ley P, Steeghs L, Hamstra HJ, ten Hove J, Zomer B, van Alphen L. Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity. Infect Immun 2001;69(10):5981-5990.

10. Steeghs L, Tommassen J, Leusen JH, van de Winkel JG, van der Ley P. Teasing apart structural determinants of ‘toxicity’ and ‘adjuvanticity’: implications for meningococcal vaccine development. J Endotoxin Res 2004;10(2):113-119.

11. Mogensen TH, Paludan SR, Kilian M, Ostergaard L. Two Neisseria meningitidis strains with different ability to stimulate Toll-like receptor 4 through the MyD88-independent pathway. Scand J Immunol 2006;64(6):646-654.

12. van der Ende A, Hopman CT, Dankert J. Deletion of porA by recombination between clusters of repetitive extragenic palindromic sequences in Neisseria meningitidis. Infect Immun 1999;67(6):2928-2934.



15. El Hamidi A, Tirsoaga A, Novikov A, Hussein A, Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization. J Lipid Res 2005;46(8):1773-1778.

16. Wilm M, Mann M. Analytical properties of the nanoelectrospray ion source. Anal Chem 1996;68(1):1-8.

17. Heckenberg SG, de Gans J, Brouwer MC et al. Clinical Features, Outcome, and Meningococcal Genotype in 258 Adults With Meningococcal Meningitis: A Prospective Cohort Study. Medicine (Baltimore) 2008;87(4):185-192.

18. Munford RS. Sensing gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun 2008;76(2):454-465.

19. Montminy SW, Khan N, McGrath S et al. Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response. Nat Immunol 2006;7(10):1066-1073.

20. Smirnova I, Mann N, Dols A et al. Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility. Proc Natl Acad Sci U S A 2003;100(10):6075-6080.

21. van der Ende A, Hopman CT, Zaat S, Essink BB, Berkhout B, Dankert J. Variable expression of class 1 outer membrane protein in Neisseria meningitidis is caused by variation in the spacing between the -10 and -35 regions of the promoter. J Bacteriol 1995;177(9):2475-2480.

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22. Hammerschmidt S, Hilse R, van Putten JP, Gerardy-Schahn R, Unkmeir A, Frosch M. Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element. EMBO J 1996;15(1):192-198.

23. Weber MV, Claus H, Maiden MC, Frosch M, Vogel U. Genetic mechanisms for loss of encapsulation in polysialyltransferase-gene-positive meningococci isolated from healthy carriers. Int J Med Microbiol 2006;296(7):475-484.

24. Meszaros K, Aberle S, Dedrick R et al. Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein. Blood 1994;83(9):2516-2525.

25. Li A, Chang AC, Peer GT, Hinshaw LB, Taylor FB, Jr. Comparison of the capacity of rhTNF-alpha and Escherichia coli to induce procoagulant activity by baboon mononuclear cells in vivo and in vitro. Shock 1996;5(4):274-279.

26. Drake TA, Cheng J, Chang A, Taylor FB, Jr. Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol 1993;142(5):1458-1470.

27. Bjerre A, Ovstebo R, Kierulf P, Halvorsen S, Brandtzaeg P. Fulminant meningococcal septicemia: dissociation between plasma thrombopoietin levels and platelet counts. Clin Infect Dis 2000;30(4):643-647.

28. Levi M, van der Poll T, Buller HR. Bidirectional relation between inflammation and coagulation. Circulation 2004;109(22):2698-2704.

29. Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986;163(3):740-745.

30. van der Poll T, de Jonge E, Levi M. Regulatory role of cytokines in disseminated intravascular coagulation. Semin Thromb Hemost 2001;27(6):639-651.

31. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 2004;5:86.

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Chapter 4

Adjunctive dexamethasone in adults with meningococcal

meningitis

Sebastiaan G.B. Heckenberg*

Matthijs C. Brouwer*

Arie van der Ende



Neurology, 2012;79(15):1563-69

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Abstract

We evaluated the implementation and effectiveness of adjunctive

dexamethasone in adults with meningococcal meningitis. We compared

2 Dutch prospective nationwide cohort studies on community-acquired

meningococcal meningitis. A total of 258 patients with CSF culture-

proven meningitis were enrolled between 1998 and 2002, before routine

dexamethasone therapy was introduced, and 100 patients from March 2006 to

January 2011, after guidelines recommended dexamethasone.

Dexamethasone was administered in 43 of 258 (17%) patients in the 1998–

2002 cohort and in 86 of 96 (90%) patients in the 2006–2011 cohort (p < 0.001),

and was started with or before the first dose of antibiotics in 12 of 258 (5%)

and 85 of 96 (89%) patients (p < 0.001). Rates of unfavorable outcome were

similar between cohorts (12 of 100 [12%] vs 30 of 258 [12%]; p = 0.67), also

after correction for meningococcal serogroup. The rates of hearing loss (3 of 96

[3%] vs 19 of 237 [8%]; p = 0.10) and death (4 of 100 [4%] vs 19 of 258 [7%]; p =

0.24) were lower in the 2006–2011 cohort, but this did not reach significance.

The rate of arthritis was lower in patients treated with dexamethasone (32 of

258 [12%] vs 5 of 96 [5%], p = 0.046). Dexamethasone was not associated with

adverse events.

Adjunctive dexamethasone is widely prescribed for patients with

meningococcal meningitis and is not associated with harm. The rate of arthritis

has decreased after the implementation of dexamethasone. This study provides

Class III evidence that adjuvant dexamethasone in adults with meningococcal

meningitis does not increase negative outcomes such as deafness, death, or

negative Glasgow Outcome Scale measures.

Introduction

Bacterial meningitis remains an important cause of morbidity and mortality

worldwide, even though effective antibiotic therapy is available and

vaccination strategies have been implemented.1-3 The most common causes

are Streptococcus pneumoniae and Neisseria meningitidis, accounting for 85%

of cases in adults.1-3 Fatality rates in patients with meningitis caused by these

microorganisms are significant, with rates of 10% and 26%,1, 4, 5 and of surviving

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patients, 30% to 50% have neurologic deficits including hearing loss6 and

cognitive deficits.7, 8

Several adjunctive therapies have been evaluated in bacterial meningitis.9,

10 Randomized clinical trials have evaluated the efficacy of adjunctive

corticosteroid therapy.11, 12 A large European randomized controlled trial in

adults showed a beneficial effect of dexamethasone in bacterial meningitis, with

the benefit most apparent in pneumococcal meningitis patients.13 Following

this study, adjunctive dexamethasone has been incorporated in treatment

guidelines in the Netherlands for pneumococcal meningitis.14 By comparing

two nationwide cohort studies (one performed before routine dexamethasone

therapy was implemented and the second one after implementation), we have

shown that the prognosis of pneumococcal meningitis on a national level

substantially improved after the implementation of dexamethasone.14

Concerns have been raised on the safety of adjunctive dexamethasone

treatment in meningitis due to other causes than S. pneumoniae, of which

meningococcal meningitis is the largest group.15, 16 Therefore, guidelines

recommend cessation of dexamethasone if pathogens other than the

pneumococcus are cultured and others recommend dexamethasone only

in patients with pneumococcal meningitis.15-17 We address these concerns,

whether side effects of dexamethasone occurred and if outcome has changed

since implementation.

Methods

We identified adults (>16 years of age) with meningococcal meningitis defined

by positive cerebrospinal (CSF) culture and were listed in the database of the

Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM) at the

Academic Medical Center, Amsterdam from March 2006 to January 2011. This

laboratory receives CSF isolates from approximately 85% patients with bacterial

meningitis, with a delay of 2 to 6 days after admission. Physicians were informed

about the study by telephone. Physicians could also contact investigators 24/7

to include patients, without preceding report of the NRLBM. Patients or their

legal representatives received written information concerning the study and

were asked to give written informed consent for participation. Online case

record forms were used to collect data. Patients with negative CSF cultures

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64

or hospital-associated meningitis were excluded. Patients with an altered

immune status owing to the use of immunosuppressive drugs or splenectomy,

diabetes mellitus, alcoholism or HIV were considered immunocompromised.

Complications that could be attributed to use of dexamethasone were scored

for both cohorts. Outcome was graded according to the Glasgow Outcome

Scale (GOS). A score of 1 on this scale indicates death; a score of 2 a vegetative

state (the patient is unable to interact with the environment); a score of 3

severe disability (the patient is unable to live independently but can follow

commands); a score of 4 moderate disability (the patient is capable of living

independently but unable to return to work or school); and a score of 5 mild

or no disability (the patient is able to return to work or school). A favorable

outcome was defined as a score of 5, and an unfavorable outcome as a score

of 1 to 4. The GOS is a well-validated instrument with good interobserver

agreement.18 At discharge, all surviving patients underwent a neurologic

examination performed by a neurologist which included the assessment of

the GOS. We compared our results with historical controls from a study with

similar design that included 258 patients with meningococcal meningitis from

1998 through 2002, before guidelines recommended routine dexamethasone

therapy.1,4 Serogrouping, susceptibility testing and multi-locus sequence

typing (MLST) of meningococcal isolates for both cohorts was performed

by the NRLBM. The Mann-Whitney U test (continuous variables) and chi2 test

(categorical variables) were used to identify differences in demographic and

clinical characteristics between the 2 cohorts.

The primary research question was if the introduction of adjunctive

dexamethasone in the Netherlands has changed outcome in meningococcal

meningitis. The study design provides a Class III level of evidence.

Results

In total, 920 from 1,119 (82%) identified episodes of community-acquired

bacterial meningitis were included in the cohort in the period 2006–2011

(figure). N meningitidis was cultured from CSF in 100 of 920 episodes (11%).

The mean age of these 100 patients with meningococcal meningitis was 38

years, and half of the patients had symptoms less than 24 hours (table 1).

Predisposing conditions were present in 18 of 100 patients (18%) and mostly

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consisted of an immunocompromised state. A relatively low number of patients

had fever (62 of 99 [62%]) or an altered mental status, defined as a score on the

Glasgow Coma Scale below 14 (50 of 99 [50%]). Therefore, the classic triad of

bacterial meningitis (neck stiffness, fever, and altered mental status) was noted

in only 27 of 98 patients (27%). At presentation, signs of septic shock (defined

as diastolic blood pressure <60 mm Hg, systolic blood pressure <90 mm Hg, or

heart rate >120/min) were present in 18 of 96 (19%) evaluated patients. A skin

rash was present in 46 of 96 (48%) patients and classified as petechial in 37,

purpura or ecchymosis in 5, and a combination of both in 4.

Figure. Selection of patients

or a white cell count of more than 2,000 per mm3)19

was present in 82 of 100 episodes (82%). Five pa-tients had fewer than 50 CSF leukocytes per mm3,and all 5 had normal CSF protein levels. CSF Gramstain showed Gram-negative diplococci in 3 of thesepatients. On admission, clinical characteristics andresults of laboratory tests between cohorts were simi-lar, although more patients admitted between 2006and 2011 were immunocompromised (13 of 100[13%] vs 10 of 258 [4%], p � 0.003) on admissionand less had a skin rash (46 of 96 [48%] vs 164 of256 [64%]; p � 0.005).

Initial antimicrobial treatment consisted ofpenicillin or amoxicillin in 31 of 96 (32%)episodes, third-generation cephalosporin in 38(39%), and a combination of penicillin or amoxi-cillin and third-generation cephalosporin in 27(28%) episodes; another regimen was used in 2patients (2%). Antibiotic susceptibility was testedin 92 strains; 87 strains were sensitive to penicillin,and 5 strains showed intermediate susceptibility topenicillin (MIC �0.094). The rate of intermediatesusceptibility to penicillin was higher compared tothe 1998–2002 cohort (5% vs 1.6%; p � 0.06). Onepatient infected with intermediate susceptible strainswas treated with microbiologically inadequate initialantimicrobial therapy (penicillin monotherapy),which was changed to a third-generation cephalo-sporin on day 5. Outcome was favorable in all 5patients infected with a meningococcal strain withreduced susceptibility.

Adjunctive dexamethasone was administered in90% of episodes (table 2), and started before or with

the first dose of antibiotics in 85 episodes (89%).Dexamethasone, 10 mg IV, given every 6 hours for 4days was started before or with the first dose of par-enteral antibiotics in 78 of 96 episodes (81%). In 6patients (6%), dexamethasone was discontinued aftercultures grew meningococci. Dexamethasone wasprescribed in 35 of 39 patients (90%) with a rash onadmission. Adjunctive dexamethasone was adminis-tered in 43 episodes (17%) in the 1998–2002 co-hort. Twelve of these patients were included in theEuropean dexamethasone in adulthood bacterialmeningitis study and received dexamethasone 10 mgIV, given every 6 hours for 4 days, started before orwith first dose of parenteral antibiotics; dexametha-sone was initiated after clinical deterioration in allother episodes.4,13

The serogrouping result was available for 90 me-ningococcal strains (table 3). Of these, 75 (83%)were of serogroup B, 7 (8%) of serogroup C, 5 (6%)of serogroup Y, 2 (2%) of serogroup W135, and 1(1%) of serogroup X. The incidence of meningococ-cal meningitis has decreased sharply between the 2cohorts. The strong decrease in serotype C meningi-tis (p � 0.001) shows vaccination programs takingfull effect during the second cohort study. However,the absolute incidence of serogroup B infection alsodeclined from 0.67 per 100,000 per year to 0.17 per100,000 per year (p � 0.001). MLST was per-formed in 89 of 100 strains (89%). The most com-mon clonal complexes (cc) were cc41/44 (34 of 89[38%]) and cc32 (19 of 89 [21%]). A marked de-crease was found for cc11 (8 of 89 [9%]) com-pared to the previous cohort study (61 of 254

Figure Selection of patients

Neurology 79 October 9, 2012 1565

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Table 1.Characteristics of Dutch adults with meningococcal meningitis in 2 nationwide cohort studiesa

Characteristic 2006-2011100 Episodes

1998-2002258 Episodes

P-value

Age - year (mean±SD) 38±19 36±19 0.44

Male sex 54 (54%) 133 (52%) 0.67

Duration of symptoms longer than 24 hours 54/99 (54%) 123/251 (51%) 0.35

Pretreated with antibiotics 5/100 (5%) 6/257 (2%) 0.19

Predisposing conditions

Otitis or sinusitis 1/100 (1%) 9/258 (4%) 0.20

Pneumonia 4/100 (4%) 13/258 (5%) 0.67

Immunocompromiseb 13/100 (13%) 10/258 (4%) 0.001

Symptoms on presentation

Headache 86/95 (91%) 223/247 (90%) 0.95

Neck stiffness 79/98 (81%) 226/255 (89%) 0.08

Heart rate ≥120 beats per minute 8/94 (9%) 17/240 (7%) 0.66

Rash 46/96 (48%) 164/256 (64%) 0.005

Body temperature >38ºC 62/99 (62%) 161/250 (64%) 0.76

Diastolic blood pressure <60 mm Hg 12/96 (13%) 37/246 (15%) 0.55

Cranial nerve palsy 4/96 (4%) 18/258 (7%) 0.33

Focal cerebral deficits 12/100 (12%) 32/258 (12%) 0.91

Score on Glasgow Coma Scale (means ±SD)c 12±3 12±3 0.50

<14 (indicating altered mental status) 50/99 (50%) 131/257 (51%) 0.94

<8 (indicating coma) 9/99 (9%) 19/257 (7%) 0.59

Triad of fever, neck stiffness and change in mental status

27/98 (27%) 70/258 (27%) 0.94

CSF findings

Opening pressure - cm H2Od 38 (27-45) 40 (22-50) 0.53

White-cell counte 5546 (1878-13500) 5328 (1590-12433) 0.55

Protein — g/litref 4.0 (1.9-6.1) 4.5 (2.2-7.0) 0.09

CSF: blood glucose ratiog 0.09 (0.00-0.35) 0.08 (0.01-0.3) 0.37

Positive gram stain 77/89 (87%) 209/244 (95%) 0.84

Blood findings

Positive blood culture 45/89 (50%) 129/227 (57%) 0.31

C-reactive protein - mg/litreh 222 (139-334) 230 (160-310) 0.76

Thrombocyte count - platelets/mm3 i 178 (137-233) 166 (126-217) 0.16aData are presented as n/N (%), continuous data are median (interquartile range). bDefined by the use of immunosuppressive drugs, a history of splenectomy, or the presence of diabetes mellitus or alcoholism, as well as patients infected with HIV. cGlasgow Coma Scale scores were evaluated in 257 patients in 1998–2002 and 99 patients in 2006–2011. dCSF pressure was measured in 92 patients in1998–2002 and in 37 patients in 2006–2011. eCSF leukocyte count was determined in 242 patients in 1998–2002 and in 95 patients in 2006–2011. fCSF protein levels were determined in 238 patients in 1998–2002 and in 92 patients in 2006–2011. g Both CSF and blood glucose values were determined in 230 patients in 1998–2002 and in 92 patients in 2006–2011. h C-reactive protein levels were determined in 150 patients in 1998–2002 and in 93 patients in 2006–2011. i Thrombocyte count was determined in patients in 1998–2002 and in 94 patients in 2006–2011.

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Cranial imaging was performed on admission in 64 patients. Abnormalities

were identified in 7 patients (brain edema in 3, hydrocephalus in 2, and

cerebral infarction and a subdural hygroma in 1 patient each). Lumbar

puncture was performed in all patients. If the lumbar puncture was preceded

by cranial imaging, antibiotics were initiated before going to the scan in only

22 of 46 (48%) episodes. At least 1 individual CSF finding predictive of bacterial

meningitis (a glucose level of less than 34 mg/dL [1.9 mmol/L], a ratio of CSF

glucose to blood glucose of less than 0.23, a protein level of more than 220

mg/dL, or a white cell count of more than 2,000 per mm3)19 was present in 82

of 100 episodes (82%). Five patients had fewer than 50 CSF leukocytes per

mm3, and all 5 had normal CSF protein levels. CSF Gram stain showed Gram-

negative diplococci in 3 of these patients. On admission, clinical characteristics

and results of laboratory tests between cohorts were similar, although more

patients admitted between 2006 and 2011 were immunocompromised (13 of

100 [13%] vs 10 of 258 [4%], p=0.003) on admission and less had a skin rash (46

of 96 [48%] vs 164 of 256 [64%]; p=0.005).

Initial antimicrobial treatment consisted of penicillin or amoxicillin in 31

of 96 (32%) episodes, third-generation cephalosporin in 38 (39%), and a

combination of penicillin or amoxicillin and third-generation cephalosporin

in 27 (28%) episodes; another regimen was used in 2 patients (2%). Antibiotic

susceptibility was tested in 92 strains; 87 strains were sensitive to penicillin, and

5 strains showed intermediate susceptibility to penicillin (MIC ≥0.094). The rate

of intermediate susceptibility to penicillin was higher compared to the 1998–

2002 cohort (5% vs 1.6%; p=0.06). One patient infected with intermediate

susceptible strains was treated with microbiologically inadequate initial

antimicrobial therapy (penicillin monotherapy), which was changed to a third-

generation cephalosporin on day 5. Outcome was favorable in all 5 patients

infected with a meningococcal strain with reduced susceptibility.

Adjunctive dexamethasone was administered in 90% of episodes (table 2),

and started before or with the first dose of antibiotics in 85 episodes (89%).

Dexamethasone, 10 mg IV, given every 6 hours for 4 days was started before

or with the first dose of parenteral antibiotics in 78 of 96 episodes (81%).

In 6 patients (6%), dexamethasone was discontinued after cultures grew

meningococci. Dexamethasone was prescribed in 35 of 39 patients (90%)

with a rash on admission. Adjunctive dexamethasone was administered in

43 episodes (17%) in the 1998–2002 cohort. Twelve of these patients were

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included in the European dexamethasone in adulthood bacterial meningitis

study and received dexamethasone 10 mg IV, given every 6 hours for 4 days,

started before or with first dose of parenteral antibiotics; dexamethasone was

initiated after clinical deterioration in all other episodes.4,13

Table 2. Characteristics of intravenous dexamethasone treatmenta


1998-2002258 Episodes

AbsoluteDifference (%)

Dexamethasone received 86 (90%) 43 (17%) +73%b

Dexamethasone 10 mg every six hours for four days, started before or with first dose of antibiotics

78 (81%) 12 (5%) +76% b

Dexamethasone started before or with first dose of antibiotics, all dosages and durations

85 (89%) 12 (5%) +84% b

Dexamethasone 10 mg every six hours for four days, started at any time

79 (82%) 12 (5%) +77% b

a Data are number of episodes (percentage).b p-value for differences between cohorts <0.001

The serogrouping result was available for 90 meningococcal strains (table 3). Of

these, 75 (83%) were of serogroup B, 7 (8%) of serogroup C, 5 (6%) of serogroup

Y, 2 (2%) of serogroup W135, and 1 (1%) of serogroup X. The incidence of

meningococcal meningitis has decreased sharply between the 2 cohorts.

The strong decrease in serotype C meningitis (p<0.001) shows vaccination

programs taking full effect during the second cohort study. However, the

absolute incidence of serogroup B infection also declined from 0.67 per 100,000

per year to 0.17 per 100,000 per year (p<0.001). MLST was performed in 89 of

100 strains (89%). The most common clonal complexes (cc) were cc41/44 (34 of

89 [38%]) and cc32 (19 of 89 [21%]). A marked decrease was found for cc11 (8

of 89 [9%]) compared to the previous cohort study (61 of 254 [24%]; p=0.004),

also caused by the vaccination for group C meningococci.

Table 3. Meningococcal serogroups

Characteristic 2006-2011 1998-2002 P-valuea

90/100 Episodes 254/258 Episodes

Serogroup B 75 (83%) 172 (68%) 0.007

Serogroup C 7 (8%) 78 (31%) <0.001

Serogroup W135 2 (2%) 1 (0.4%) 0.16

Serogroup X 1 (1%) 0 0.26

Serogroup Y 5 (6%) 3 (1%) 0.03a compared to all other serogroups

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During clinical course, neurologic complications (impairment of consciousness,

seizures, or focal neurologic abnormalities) occurred in 43 episodes (43%) and

cardiorespiratory failure in 18% (table 4). Neurologic complications were as

likely to occur in 2006–2011 as compared with 1998–2002. The rate of septic

shock between cohorts was similar as well. The rate of arthritis was significantly

lower in patients receiving adjunctive dexamethasone (5 of 96 [5%] vs 32 of

258 [12%], p= 0.046).

Few complications attributable to dexamethasone occurred, including

hyperglycemia requiring insulin (1/96 [1%] vs 1/261 [0.4%], p=0.93) and herpes

simplex infection (3/96 [3%] vs 2/261 [0.8%], p= 0.25). No gastric bleeding

occurred in the current study.

The mortality rate was 4% (table 4) and 12% of episodes had an unfavorable

outcome. Neurologic examination was performed at discharge in 96 surviving

patients and showed neurologic sequelae in 10 patients (11%). Cranial nerve

palsies were identified on discharge in 6 patients and hearing loss in 3. The

proportion of patients with unfavorable outcome (GOS score of 1 to 4) was

identical in the 2006–2011 cohort as compared to the 1998–2002 cohort

(12 of 100 [12%] vs 30 of 258 [12%]; odds ratio 1.04, 95% confidence interval

0.48–2.02, p=0.96). After correction for meningococcal serogroup the rates

of unfavorable outcome remained similar between cohorts. Outcomes of

immunocompromised patients between cohorts were similar. The rates of

hearing loss (3 of 96 [3%] vs 19 of 237 [8%]; p=0.10) and death (4 of 100 [4%]

vs 19 of 258 [7%]; p=0.24) were lower in the 2006–2011 cohort, but this did not

reach significance.

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Table 4. Clinical course, mortality, disability, and neurologic findings at dischargea


1998-2002258 Episodes

Difference (%)b

Clinical course

Neurologic complications 43 (43%) 105/258 (41%) +2%

Cardiorespiratory failure 18 (18%) 44 (17%) +1%

Score on Glasgow Outcome Scale

1 (death) 4 (4%) 19 (7%) -3% (p=0.24)

2 (vegetative state) 0 0 0

3 (severe disability) 0 4 (2%) -2%

4 (moderate disability) 8 (8%) 7 (3%) +5%

5 (no or minor disability) 88 (88%) 228 (88%) 0%

Neurologic findings at discharge

Cranial nerve palsy 6/96 (6%) 6/238 (3%) +3%

Hearing impairment 3/96 (3%) 19/237 (8%) -5% (p=0.10)

Focal cerebral deficits 4/96 (4%) 4 (2%) +2%a Data are presented as n/N (%), b p-value for all >0.05.

Discussion

Our study shows that adjunctive dexamethasone is widely prescribed in

patients with meningococcal meningitis in the Netherlands. The drug was given

to 90% of patients included between 2006-2011, which is comparable to the

prescription rate in patients with pneumococcal meningitis.14 Dexamethasone

was stopped after identification of meningococci in a small minority of patients

(6%), even though discontinuation is advised by the IDSA guideline.16 Most

patients (81%) received the recommended dose of 10mg every 6 hours for 4

days started before or with the first dose of antibiotics.

Adjunctive dexamethasone did not influence rates of unfavorable outcome.

However, there was a favorable trend for death and hearing loss in the

meningococcal subgroup in the absence of any excess adverse events. Patients

treated with adjunctive dexamethasone did not experience a higher rate of

dexamethasone related complications such as hyperglycemia requiring

insulin, gastric bleeding, and herpes simplex infection. We did observe a

reduced rate of autoimmune mediated arthritis, which provides an argument

in favor of dexamethasone treatment. Arthritis is a common manifestation in

patients with community-acquired bacterial meningitis.20 Functional outcome

of arthritis in bacterial meningitis is generally good because meningococcal

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arthritis is usually immune-mediated. To show a similar reduction in mortality

as found in pneumococcal meningitis (relative risk reduction 33%, absolute

risk reduction 10%),14 1488 patients with meningococcal meningitis have to

be included in a randomized controlled trial (power 0.80, α 0.05). Currently, it

seems unlikely that a study will be performed with enough power to prove or

disprove an effect of adjunctive dexamethasone treatment on meningococcal

meningitis.

Hospitals will require protocols to include dexamethasone with initial antibiotic

therapy, since the causative organism will not be know in many cases when

treatment is begun. In the Netherlands, physicians do not appear to differentiate

between possible causative agents in prescribing dexamethasone since the

majority of patients with rash, known as a typical sign of meningococcal

disease, received adjunctive dexamethasone. As we observed no excess of

dexamethasone related complications, safety concerns on dexamethasone in

meningococcal meningitis should not prevent treatment of suspected bacterial

meningitis patients with adjunctive dexamethasone. Based on our results there

is no need to discontinue empiric treatment with adjunctive dexamethasone

in patients with culture-proven meningococcal meningitis.

Our study has limitations. The observational design of the study is sensitive

to the introduction of confounding factors, which hinder the evaluation of

dexamethasone effectiveness. Observed differences between cohorts such

as rate of immunocompromise or rash may disturb the comparison between

cohorts. An important difference between cohorts was the decline in incidence

of meningococcal meningitis in general, and the sharp decline of meningitis

due to group C meningococci compared to group B. This is caused by the

introduction of the vaccine against group C meningococci in the Netherlands

in 2002, and also explains the shift in clonal complex frequency. Correction

for meningococcal serogroup did not influence the lack of differences in

outcome between cohorts. Due to the low event rate in both cohorts (12%

unfavorable outcome) and decrease in incidence our study lacked power to

perform a multivariate adjustment for differences in case mix between cohorts.

Patients presenting with meningococcal meningitis and septic shock are

underrepresented in our cohorts, as lumbar puncture is often deferred in these

patients. In patients with septic shock high dose steroids are not beneficial and

therefore dexamethasone should be withheld in these patients.21

Despite these limitations of the study design we think this study provides

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valuable information on the use of dexamethasone in meningococcal meningitis.

Although we did not show any differences in unfavorable outcome, there was

a favorable trend for death and hearing loss in the meningococcal subgroup in

the absence of any excess adverse events. Therefore, dexamethasone can be

safely administered in all patients suspected for community acquired bacterial

meningitis. When the patient is identified to have meningococcal meningitis

there is no obvious reason to discontinue dexamethasone.

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References

1. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351: 1849-59.

2. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53.

3. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 2010;23:467-92.

4. Heckenberg SG, de Gans J, Brouwer MC et al. Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-92.

5. Weisfelt M, van de Beek D, Spanjaard L, Reitsma JB, de Gans J. Clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series. Lancet Neurol 2006;5:123-9.

6. Heckenberg SG, Brouwer MC, van der Ende A, Hensen EF, van de Beek D. Hearing loss in adults surviving pneumococcal meningitis is associated with otitis and pneumococcal serotype. Clin Microbiol Infect 2012;18(9):849-55.

7. van de Beek D, Schmand B, de Gans J et al. Cognitive impairment in adults with good recovery after bacterial meningitis. J Infect Dis 2002;186:1047-52.

8. Hoogman M, van de Beek D, Weisfelt M, de Gans J, Schmand B. Cognitive outcome in adults after bacterial meningitis. J Neurol Neurosurg Psychiatry 2007;78:1092-6.

9. van de Beek D, Weisfelt M, de Gans J, Tunkel AR, Wijdicks EF. Drug Insight: adjunctive therapies in adults with bacterial meningitis. Nat Clin Pract Neurol 2006;2:504-16.

10. Mook-Kanamori BB, Geldhoff M, van der Poll T, van de Beek D. Pathogenesis and pathophysiology of pneumococcal meningitis. Clin Microbiol Rev 2011;24:557-91.


12. van de Beek D, Farrar JJ, de Gans J et al. Adjunctive dexamethasone in bacterial meningitis: a meta-analysis of individual patient data. Lancet Neurol 2010;9:254-63.

13. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-56.

14. Brouwer MC, Heckenberg SG, de Gans J, Spanjaard L, Reitsma JB, van de Beek D. Nationwide implementation of adjunctive dexamethasone therapy for pneumococcal meningitis. Neurology 2010;75(17):1533-1539.

15. Tunkel AR, Scheld WM. Corticosteroids for everyone with meningitis? N Engl J Med 2002; 347:1613-5.

16. Tunkel AR, Hartman BJ, Kaplan SL et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39:1267-84.

17. Chaudhuri A, Martinez-Martin P, Kennedy PG et al. EFNS guideline on the management of community-acquired bacterial meningitis: report of an EFNS Task Force on acute bacterial meningitis in older children and adults. Eur J Neurol 2008;15:649-59.

18. Jennett B, Teasdale G, Braakman R, Minderhoud J, Knill-Jones R. Predicting outcome in individual patients after severe head injury. Lancet 1976;1:1031-4.

19. Spanos A, Harrell FE, Jr., Durack DT. Differential diagnosis of acute meningitis. An analysis of the predictive value of initial observations. JAMA 1989;262:2700-7.

20. Weisfelt M, van de Beek D, Spanjaard L, de Gans J. Arthritis in adults with community-acquired bacterial meningitis: a prospective cohort study. BMC Infect Dis 2006;6:64.

21. Annane D, Bellissant E, Bollaert PE et al. Corticosteroids in the treatment of severe sepsis and septic shock in adults: a systematic review. JAMA 2009;301:2362-75.

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Chapter 5

Nationwide evaluation of implementation and

effectiveness of adjunctive dexamethasone in adult

pneumococcal meningitis


Sebastiaan G.B. Heckenberg*

Jan de Gans

Lodewijk Spanjaard

Johannes B. Reitsma



Neurology, 2010;75(17):1533-9

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Abstract

In this nationwide prospective cohort study we evaluated the implementation

and effectiveness of adjunctive dexamethasone therapy in Dutch adults with

pneumococcal meningitis.

From March 2006 through January 2009, all Dutch patients over 16 years

old with community-acquired pneumococcal meningitis were prospectively

evaluated. Outcome was classified as unfavorable (defined by a Glasgow

Outcome Scale score of 1 to 4 points at discharge) or favorable (a score of 5).

Clinical characteristics and outcome were compared with a similar nation-wide

cohort of 352 patients with pneumococcal meningitis from a previous period

before guidelines recommended dexamethasone therapy (1998-2002). A

multivariable prognostic model was used to adjust for differences in case-mix

between the two cohorts.

We evaluated 357 episodes with pneumococcal meningitis in 2006-2009.

Characteristics on admission were comparable with the earlier cohort (1998-

2002). Dexamethasone was started with or before the first dose of antibiotics in

84% of episodes in 2006-2009 and 3% in 1998-2002. At discharge, unfavorable

outcome was present in 39% in 2006-2009 and 50% in 1998-2002 (odds ratio,

0.63; 95% confidence interval, 0.46 to 0.86; p=0.002). Rates of death (20% vs.

30%; p=0.001) and hearing loss (12% vs. 22%; p=0.001) were lower in 2006-

2009. Differences in outcome remained after adjusting for differences in case-

mix between cohorts.

In conclusion, dexamethasone therapy has been implemented on a large

scale as adjunctive treatment of adults with pneumococcal meningitis in the

Netherlands. The prognosis of pneumococcal meningitis on a national level has

substantially improved after the introduction of adjunctive dexamethasone

therapy suggesting a causal effect.

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Background

In 2004, we published a nationwide prospective cohort study in adults with

bacterial meningitis from 1998 through 2002.1 In this study, 696 adults with

community-acquired bacterial meningitis confirmed by cerebrospinal fluid

culture were included and most common pathogens were Streptococcus

pneumoniae (51%) and Neisseria meningitidis (37%). The mortality rate was 21%

and half of surviving patients had neurologic sequelae.1, 2 The mortality rate

was highest among patients with pneumococcal meningitis (30%). The study

was performed before routine dexamethasone therapy was introduced.1

Experimental models have shown that treatment with corticosteroids resulted in a

reduction of the inflammatory response in the subarachnoid space and improved

outcome.3, 4 In 2002, a European multicenter randomized clinical trial showed a

beneficial effect of adjunctive dexamethasone therapy in adults with bacterial

meningitis.5 In this clinical trial, treatment with dexamethasone was associated

with a reduction in the risk of an unfavourable outcome (relative risk 0.59; 95%

confidence interval [CI] 0.37– 0.94; p=0.03). The effect was most apparent in the

pneumococcal subgroup (relative risk 0.50; 95% CI 0.30–0.83; p=0.006).

Four large randomized clinical trials on adjunctive dexamethasone in bacterial

meningitis showed conflicting results.6–9 A recent meta-analysis of individual

patient data of 5 recent randomized controlled trials showed no effect of

adjunctive dexamethasone in pneumococcal meningitis.10 Therefore, the

use of dexamethasone in pneumococcal meningitis remains controversial.

Nevertheless, guidelines recommend routine use of adjunctive dexamethasone

in adults with pneumococcal meningitis in high-income countries.11–13 We

assessed the implementation of adjunctive dexamethasone therapy in adults

with pneumococcal meningitis and its impact on outcome.

Methods

We identified adults (defined as patients older than 16 years of age) who had

pneumococcal meningitis defined by positive CSF culture and were listed in

the database of the Netherlands Reference Laboratory for Bacterial Meningitis

from March 2006 to January 2009. This laboratory receives CSF isolates from

approximately 85% of all patients with bacterial meningitis in the Netherlands

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(population, 16.2 million).14 Daily updates were provided of hospitals where

patients with bacterial meningitis had been admitted in the preceding 2 to

6 days. Physicians were informed about the study by telephone. Physicians

could also contact investigators 24/7 to include patients, without preceding

report of the reference laboratory. Patients or their legal representatives received

written information concerning the study and were asked to give written

informed consent for participation. Online case record forms were used to collect

data. Patients with negative CSF cultures or hospital-associated meningitis

were excluded. Patients with an altered immune status owing to the use of

immunosuppressive drugs or splenectomy, diabetes mellitus, or alcoholism

were considered immunocompromised, as were patients infected with HIV.

Outcome was graded according to the Glasgow Outcome Scale. A score of 1 on

this scale indicates death; a score of 2 a vegetative state (the patient is unable

to interact with the environment); a score of 3 severe disability (the patient is

unable to live independently but can follow commands); a score of 4 moderate

disability (the patient is capable of living independently but unable to return

to work or school); and a score of 5 mild or no disability (the patient is able to

return to work or school). A favorable outcome was defined as a score of 5, and

an unfavourable outcome as a score of 1 to 4. The Glasgow Outcome Scale is

a well-validated instrument with good interobserver agreement.15 At discharge,

all surviving patients underwent a neurologic examination performed by a

neurologist which included the assessment of the Glasgow Outcome Scale.

We compared our results with historical controls from a study with similar design

that included 352 patients with pneumococcal meningitis from 1998 through

2002, before guidelines recommended routine dexamethasone therapy.1, 2

The Mann-Whitney U test (continuous variables) and χ²-test (categorical

variables) were used to identify differences in demographic and clinical

characteristics between the 2 cohorts. In the earlier cohort we developed

a prediction model with 18 potentially relevant prognostic factors for

unfavorable outcome. We used logistic regression analysis to calculate odds

ratios (ORs) and 95% CIs to assess the strength of the association between

potential prognostic factors and the probability of an unfavorable outcome.

Missing values were imputed by use of multivariate normal distributions and

coefficients of 10 rounds of imputation were combined to obtain the final

estimates from the multivariate logistic regression model. The coefficients of

the multivariable prediction model were applied to obtain a risk score for each

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patient in the recent cohort. This risk score incorporates all available data on risk

factors given an individual profile on clinical and demographic characteristics.

We used these risk scores to calculate the expected number of events (if

dexamethasone would not have been introduced) and compared these with

the actually observed number of patients with unfavourable outcome in the

recent cohort. Expected vs observed numbers were calculated for the recent

cohort as a whole, across tertiles of predicted risk (i.e., severity of disease), and

for various clinical subgroups. All statistical tests were 2-tailed, and a p value

less than 0.05 was regarded as significant. Analyses were undertaken with SAS

software version 9.1.

The primary research question was if the introduction of adjunctive

dexamethasone in the Netherlands has improved outcome in pneumococcal

meningitis. The study design provides a Class III level of evidence.

Standard protocol approvals, registrations, and patient consents.

The study was approved by all Dutch local ethics committees. All participating

hospitals and local investigators are presented in appendix 5.1.

Results

A total of 787 episodes of bacterial meningitis were identified from March

2006 through January 2009 (figure 1). The cohort consisted of 518 episodes

of community-acquired bacterial meningitis, including 357 episodes of

pneumococcal meningitis in 354 patients. Classic symptoms and signs of

meningitis were present in a large proportion of the patients (table 1). The

classic triad of neck stiffness, fever, and altered mental status (defined as a

score on the Glasgow Coma Scale _14) was present in 54% and coma (defined

as a score on the Glasgow Coma Scale <8) in 18%. At least one individual CSF

finding predictive of bacterial meningitis (a glucose level of less than 34 mg/

dL [1.9 mmol/L], a ratio of CSF glucose to blood glucose of less than 0.23, a

protein level of more than 220 mg/dL, or a white-cell count of more than 2,000

per mm3)16 was present in 328 of 348 episodes (94%). At admission, clinical

characteristics and results of laboratory tests between cohorts were similar,

although more episodes had positive blood cultures (85% vs 74%; p=0.002)

and less episodes of cranial nerve palsies (7% vs 12%; p=0.02) in 2006–2009.

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The proportions of patients included in the 8 Dutch academic hospitals were

similar between 2 time periods (11.7% vs 11.8%; p=0.96).

Cranial CT was performed on admission in 320 episodes (90%); results were

normal in 52%. Abnormalities found were mastoid or sinus opacification in

37%, generalized brain edema in 18%, recent brain infarction in 7%, and other

abnormalities in 16%. Imaging preceded lumbar puncture in 251 episodes

(78%). The proportion of patients in the 2006–2009 cohort who experienced a

delay in therapy due to cranial CT was unchanged compared to patients from

1998 to 2002 (155 of 357 [43%] vs 149 of 352 [42%]; p=0.83).

Figure 1. Selection of patients

Antimicrobial treatment consisted of penicillin or amoxicillin in 33% of episodes,

third-generation cephalosporins in 28%, and a combination of penicillin

or amoxicillin and third-generation cephalosporins in 34% of episodes;

another regimen was used in 5%. Antibiotic treatment was in compliance

with Dutch guidelines in 33% of episodes. The Dutch guideline recommends

empirical therapy consisting of penicillin for adults between 16 and 60 years

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old, and empirical therapy consisting of amoxicillin plus a third-generation

cephalosporin for patients over 60 years old or those with risk factors (defined

as altered immune status, alcohol abuse, CSF leak, or recent head trauma).17

Guidelines for antibiotic use for meningitis in the Netherlands have not been

changed from 1998 to 2009. Adherence to antibiotic guidelines between

cohorts was similar (cohort 1998–2002, 32%).17

Antibiotic susceptibility testing was performed in 327 episodes; 2 strains

showed intermediate susceptibility to penicillin (MIC 1.0 mg/L and 0.125 mg/L).

The most common serotypes were type 3 and 14 (each 10%; supplementary

table 5.1); type 19F, 7F, and 9V (each 8%); and type 6B and 10A (each 6%).

The proportions of disease due to strains that were covered by the 7-valent

pneumococcal conjugate vaccine (PCV7) serotypes were unchanged in 2006–

2009, compared to 1998–2002 (42% in 2006–2008 vs 38% in 1998–2002;p=0.28).

Adjunctive dexamethasone was administered in 92% of episodes (table 2).

Dexamethasone, 10 mg IV, given every 6 hours for 4 days was started before

or with the first dose of parenteral antibiotics in 276 of 357 episodes (77%).

Dexamethasone was given after the first dose of antibiotics in 28 episodes

(8%); in 3 episodes this was prompted by clinical deterioration. Clinical

deterioration in these 3 patients was caused by brain edema in 2 patients and

respiratory failure due to bronchiolitis obliterans organizing pneumonia in 1

patient. There were no differences between patients treated with or without

early dexamethasone with respect to antibiotic pretreatment (14% vs 11%;

p=0.79), immunocompromised state (22% vs 29%; p=0.31), diastolic blood

pressure (median, 80 vs 80 mm Hg; p=0.61), or heart rate (median, 100 vs 100

beats per minute; p=0.77). Adjunctive dexamethasone was administered in

59 episodes (17%) in 1998–2002. Eleven of these patients were included in

the European dexamethasone in adulthood bacterial meningitis study and

received dexamethasone 10 mg IV, given every 6 hours for 4 days, started

before or with first dose of parenteral antibiotics; dexamethasone was initiated

after clinical deterioration in all other episodes.5

During clinical course, neurologic complications (impairment of consciousness,

seizures, or focal neurologic abnormalities) occurred in 60% of episodes and

cardiorespiratory failure in 37% (table 3). Neurologic complications, including

epileptic seizures, were less likely to occur in 2006–2008 as compared with

1998–2002 (60% vs 75%; p=0.001). The rate of cardiorespiratory failure between

cohorts was similar.

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Table 1. Characteristics of Dutch adults with pneumococcal meningitis in two nationwide cohort studiesa

Characteristic 2006-2009 357 Episodes 1998-2002 352 Episodes

Age - year (means ±SD) 59±15 58±17

Male sex 167/357 (47) 171/352 (49)

Duration of symptoms longer than 24 hours 177/343 (52) 162/362 (50)

Pretreated with antibiotics 40/344 (11) 44/349 (13)


Otitis or sinusitis 145/357 (41) 153/352 (43)

Pneumonia 56/349 (16) 62/352 (18)

Immunocompromiseb 83/357 (23) 76/351 (22)

Remote head injury 14/357 (4) 19/352 (5)

CSF leak 7/357 (2) 11/352 (3)

Symptoms on presentation

Headache 259/305 (85) 256/305 (84)

Neck stiffness 260/340 (76) 280/344 (81)

Heart rate ≥120 beats per minute 70/353 (20) 84/331 (25)

Body temperature >38ºC 285/354 (81) 291/345 (84)

Diastolic blood pressure <60 mm Hg 33/354 (9) 18/342 (5)

Score on Glasgow Coma Scale, mean±SD c 10±3 10±3

<14 (indicating altered mental status) 289/357 (81) 298/351 (85)

<8 (indicating coma) 65/357 (18) 68/351 (19)

Triad of fever, neck stiffness, and change in mental status

188/347 (54) 206/352 (59)


Aphasia 63/186 (34) 79/234 (34)

Hemiparesis 39/310 (13) 39/344 (11)

Cranial nerve palsy (excluding hearing loss)

24/346 (7) 43/352 (12) d

Hearing loss 6/346 (2) 23/243 (9) d

CSF findings

Opening pressure - cm H2Oe 42 (30-50) 40 (25-50)

White-cell countf 2490 (512-7733) 2530 (531-6983)

Protein — g/Lg 4.1 (2.5-6.1) 4.7 (2.7-7.0)

CSF: blood glucose ratioh 0.02 (0.00-0.16) 0.06 (0.01-0.20)

Positive gram stain 330/342 (96) 304/327 (93)

Blood findings

Positive blood culture 261/308 (85) 230/309 (74)

C-reactive protein - mg/litre i 215 (104-335) 211 (104-333)

Thrombocyte count - platelets/mm3lj 200 (151-262) 199 (157-250)a Data are number/number assessed (percent) or median (25th-75th percentile), unless otherwise stated. b Immunocompromise was defined by the use of immunosuppressive drugs, a history of splenectomy, or the presence of diabetes mellitus or alcoholism, as well as patients infected with HIV. c Glasgow Coma Scale scores were evaluated in 351 patients in 1998-2002 and 357 patients in 2006-2009. d p-value for difference between groups <0.05.

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e CSF pressure was measured in 114 patients in 1998-2002 and in 117 patients in 2006-2009. f CSF leukocyte count was determined in 320 patients in 1998-2002 and in 343 patients in 2006-2009. g CSF protein levels were determined in 316 patients in 1998-2002 and in 342 patients in 2006-2009. h Both CSF and blood glucose values were determined in 309 patients in 1998-2002 and in 341 patients in 2006-2009. i C-reactive protein levels were determined in 187 patients in 1998-2002 and in 331 in cohort 2006-2009. j Thrombocyte count was determined in 326 patients in 1998-2002 and in 344 patients in cohort 2006-2009.

Table 2. Characteristics of intravenous dexamethasone treatmenta


1998- 2002 352 Episodes

Absolute difference (%)

Dexamethasone received 329 (92%) 59 (17%) +75%b

Dexamethasone 10 mg every 6 hours for 4 days, started before or with first dose of antibiotics

276 (77%) 11 (3%) +74%b

Dexamethasone started before or with first dose of antibiotics, all dosages and durations

301 (84%) 11 (3%) +81%b

Dexamethasone 10 mg every six hours for 4 days, started at any time

299 (84%) 11 (3%) +81%b

a Data are number of episodes (percentage). b p-value for differences between cohorts <0.001

The mortality rate was 20% (table 3) and 39% of episodes had an unfavorable

outcome. Neurologic examination was performed at discharge in 280 of

285 surviving patients (98%); most common abnormalities were hearing

impairment (12%) and focal cerebral deficits (11%). The proportion of patients

with unfavorable outcome (Glasgow Outcome Scale score of 1 to 4) was lower

in the 2006–2009 cohort, as compared to the 1998 –2002 cohort (39% vs 50%;

OR 0.63; 95% CI 0.46–0.86; p=0.002). Mortality rate (20% vs 30%; absolute risk

difference 10%; 95% CI 4%–17%; p=0.001) was also lower in 2006–2009.

On average, 3% of the values were missing and had to be imputed in the

multivariate prediction model (Supplementary table 5.2). The observed

unfavorable outcome in 2006–2009 of 39% was lower than the predicted

49% based on the multivariable prognostic model (p=0.007). Figure 2A shows

observed and predicted risks of unfavorable outcome within 3 groups of

patients with increasing disease severity. Observed numbers of patients with

unfavorable outcome in 2006–2009 were lower than the expected numbers

in the middle and high-risk groups, whereas no differences were observed in

the low-risk group. The improved outcome was primarily observed in those

patients receiving the standard regimen of dexamethasone (figure 2B). There

was no difference in observed and predicted outcome in patients who did not

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Table 3. Clinical course, mortality, disability, and neurologic findings at dischargea


1998-2002352 Episodes

Difference (%)

p-value

Clinical course

Neurologic complicationsb 239 (60%) 263 (75%) -15% <0.001

Seizures 60/344 (17%) 85/349 (24%) -7% 0.025

Cardiorespiratory failure 133 (37%) 134 (38%) -1% 0.823


1 (death) 71 (20%) 107 (30%) -10% 0.001

2 (vegetative state) 0 3 (1%) -1%

3 (severe disability) 18 (5%) 17 (5%) 0%

4 (moderate disability) 50 (14%) 50 (14%) 0%

5 (no or minor disability) 218 (61%) 175 (50%) +11% 0.002

Neurologic findings at discharge

Cranial nerve palsy 47/280 (17%) 67/243 (28%) -11% 0.003

Hearing impairment 33/280 (12%) 55/243 (22%) -10% 0.001

Focal cerebral deficits 32/280 (11%) 26/243 (11%) 0% 0.791a Neurological examination was performed in 243 of 245 surviving patients of cohort 1998-2002 and 280 of 285 surviving patients of cohort 2006-2009. b Neurologic complications were defined as impairment of consciousness, seizures, or focal neurological abnormalities.

Figure 2. Observed and predicted rates of unfavorable outcome in 2006-2009

Panel A shows predicted and observed rates of unfavorable outcome for groups with low, middle, and high risk for unfavorable outcome (groups based on tertiles). Panel B shows predicted and observed rates of unfavorable outcome for patients not treated with dexamethasone, those who received the recommended standard dexamethasone regimen (10 mg intravenously, given every six hours for four days, started before or with the first dose of parenteral antibiotics), and those who received an alternative regimen of dexamethasone. The absolute difference between predicted and observed rates of unfavorable outcome is noted above bars.

receive dexamethasone therapy. We explored the differences between

observed and predicted outcome for various clinical subgroups of patients

(Supplementary figure 5.1). The difference in observed vs predicted unfavorable

outcome rate was largest in the subgroup of patients over aged 55 years,

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those without antibiotic pretreatment, and those with high CSF protein levels

indicating severe CSF inflammation. This suggests that dexamethasone is most

effective in patients with these characteristics.

Discussion

Dexamethasone therapy has been implemented on a large scale as adjunctive

treatment of adults with pneumococcal meningitis in the Netherlands. The

drug was administered in 92% of episodes in 2006 –2009. The large majority of

physicians adhered to current guidelines recommending a standard regimen

of dexamethasone, 10 mg IV, given every 6 hours for 4 days, started before or

with the first dose of parenteral antibiotics.

The outcome of adults with community-acquired pneumococcal meningitis on

a national level has significantly improved over the last few years. We found a

decline in case fatality from 30% to 20%. This observation cannot be attributed

to a change in disease severity as we corrected for a large set of prognostic

factors. The main difference between cohorts was the successful introduction

of adjunctive dexamethasone therapy in the Netherlands. The decline in case

fatality that we observed matched the results of a randomized clinical trial that

we performed in a comparable population.5

The use of observational data in the evaluation of treatment effects raises

debates.18, 19 The greatest concern with observational studies is the issue

known as confounding by indication.20, 21 Confounding by indication refers to

the situation in daily clinical practice that prescribing will be guided by the

prognosis of the patient: the worse the prognosis, the more or stronger therapy

will be given. This means mixing treatment decisions with prognosis and that

correction for important prognostic factors may only remove part of this bias.

For several reasons, we believe that our observational data may provide valuable

evidence in the controversy about the effectiveness of dexamethasone. First,

our key analysis is based on comparing 2 national cohorts on an intention-to-

treat basis (one from a period in which hardly any dexamethasone was used,

compared to a cohort in which dexamethasone was generally prescribed). The

bias due to prescribing dexamethasone to patients who are systematically in

poorer or better condition does not apply when comparing 2 national cohorts

as a whole. Second, we applied an extensive adjustment for differences in case

mix between the 2 cohorts based on a large and independent body of data on

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prognostic factors in bacterial meningitis.1, 2 Third, there are no indications of

improvements in other (supportive) treatment options for bacterial meningitis

that could explain such a large improvement. Fourth, the treatment benefit

observed in our observational

study was similar in magnitude as reported in the randomized clinical trial

on dexamethasone. The vast majority of patients included in this trial were

Dutch patients. Finally, the benefit of dexamethasone was observed across the

whole study population, but was more prominent in patients actually receiving

dexamethasone (per-treatment analysis).

Dexamethasone appears to be more effective in patients aged older than

55 years. This is consistent with findings of a recent individual patient data

meta-analysis including 2,029 patients from 5 randomized controlled trials.10

Dexamethasone was not associated with a reduction in death in this meta-

analysis (OR 0.97, 95% CI 0.79–1.19), but was effective in patients aged older

than 55 years (OR for death 0.41 [95% CI 0.20–0.84], p=0.01). In the meta-

analysis the apparent benefit in adults aged over 55 years was interpreted as

having occurred by chance, since there was no clear evidence of heterogeneity

between the different age groups. Previous studies showed that induction of

pro-inflammatory cytokines after septic stimuli is not adequately controlled by

anti-inflammatory mechanisms in elderly persons.22 An age-related beneficial

effect of dexamethasone could be an explanation for the apparent conflicting

results of recent randomized controlled trials.5, 6, 8, 9, 23 Perhaps dexamethasone

is most effective in older patients with severe CNS inflammation without

antibiotic pretreatment.

From 1950 onwards, the introduction of modern hospital facilities,

intensive care units, cranial CT, and evidence-based guidelines may all have

contributed to the steady and gradual decrease from 40% to 30% in mortality

of community-acquired pneumococcal meningitis.24 We now observed a

further decrease in mortality of 10%, within a 4-year period, that could not

be explained by differences in case mix. This study provides Class III evidence

that dexamethasone given every 6 hours for 4 days reduced the proportion of

patients with unfavourable outcome and reduced mortality in pneumococcal

meningitis in adults. Our observation supports the use of adjunctive

dexamethasone in adult pneumococcal meningitis in high-income countries.

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Supplementary table 5.1. Capsular pneumococcal serotypes

Serotype 2006-2009 cohortN=327a

1998-2002 cohortN=352

3 31 (10%) 36 (10%)

14b 15 (5%) 34 (10%)

19Fb 13 (4%) 29 (8%)

7F 39 (12%) 28 (8%)

9Vb 9 (3%) 27 (8%)

6Bb 23 (7%) 21 (6%)

10A 10 (3%) 21 (6%)

8 27 (8%) 16 (5%)

4b 15 (5%) 16 (5%)

23Fb 34 (10%) 14 (4%)

6Ac 7 (2%) 10 (3%)

19A 5 (2%) 10 (3%)

12F 3 (1%) 10 (3%)

22F 19 (6%) 8 (2%)

18Cb 14 (5%) 8 (2%)

1 12 (4%) 5 (1%)

Otherd 51 (16%) 63 (18%)

Total PCV7 serotypes 125 (38%) 149 (42%)

PCV7-related serotypes 22 (6%) 26 (7%)a Serotypes were unavailable in 30 of 357 episodes (8%). b Serotypes included in the 7-valent pneumococcal conjugate vaccine (PCV7). c PCV7 vaccine-related serotypes d Other types in cohort 1 were as follows: in 35F in six, 9Nc in five, 17F in five, 38 in four, 15B in four, 16F in four, 18Bc in four, 33F in four, 23Bc in four, 24F in three, 34 in three, 5 in two, 15A in two, 15C in two, 20 in two, 22A in two, 9Ac in one, 18Fc in one, and 23Ac in one. Other types in cohort 2: 11A in seven, 23Bc in six, 33F in four, 31 in three, 15C in three, 16F in three, 18Bc in three, 23Ac in three, 35F in three, 9Nc in three, 20 in two, 15B in two, 24F in two, 24F in two, 25 in one, 34 in one, 38 in one, 15A in one, 17F in one.

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Supplementary table 5.2. Multivariate analysis model after imputation, cohort 1998-2002

Patient characteristic Odds ratio (95% confidence interval)

p-value Missing values

Age (per 10 year) 1.14 (0.98-1.34) 0.093 0

Duration of symptoms (>24hours) 1.53 (0.89-2.64) 0.127 26 (7%)

Seizures before admission 0.79 (0.28-2.26) 0.660 1 (0.3%)

Antibiotics before admission 0.64 (0.29-1.38) 0.251 3 (1%)

Distant focus of infectiona 1.21 (0.72-2.04) 0.465 0

Immuncompromiseb 2.03 (1.07-3.82) 0.029 1 (0.3%)

Neck stiffness 0.40 (0.20-0.81) 0.011 8 (2%)

Heart rate >120 beats per minute 1.29 (0.57-2.91) 0.542 21 (6%)

Diastolic blood pressure <60mmHg 1.89 (0.62-5.74) 0.262 8 (2%)

Temperature >38·0 °C 1.47 (0.68-3.17) 0.327 7 (2%

Score on Glasgow Coma Scale 0.90 (0.82-1.00) 0.047 1 (0.3%)

Focal cerebral deficit 1.63 (0.91-2.90) 0.098 0

Cranial nerve palsy (excluding hearing loss) 1.90 (0.95-3.81) 0.071 0

CSF leukocytes count <1000/mm3 4.74 (2.44-9.21) <0.001 32 (9%)

CSF protein (per 1g/L) 1.21 (1.09-1.35) 0.001 36 (9%)

CSF to blood glucose ratio (per 0·20) 0.81 (0.58-1.12) 0.201 43 (12%)

Blood culture 0.97 (0.50-1.87) 0.922 43 (12%)

Thrombocyte count (per 100 000) 0.84 (0.64-1.10) 0.201 26 (7%)a Defined as pneumonia, otitis or sinusitis. b Immunocompromise was defined by the use of immunosuppressive drugs, a history of splenectomy, or the presence of diabetes mellitus or alcoholism, as well as patients infected with human immunodeficiency virus (HIV).

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Supplementary figure 5.1. Explorative analysis of differences between observed and predicted outcome

Figure e-1. Panel A-D show explorative analyses of differences between observed and predicted outcome.

CSF denotes cerebrospinal fluid. The absolute difference between predicted and observed rates of

unfavorable outcome is noted above bars.

CSF denotes cerebrospinal fluid. The absolute difference between predicted and observed rates of unfavorable outcome is noted above bars.

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Supplementary methods

List of participating hospitals, local investigators (number of patients enrolled)

Academisch Medisch Centrum (27), Atrium Medisch Centrum, M.J. Wennekes

(18), Universitair Medisch Centrum Sint Radboud, R.A.J. Esselink (18), Amphia

Ziekenhuis, R.J. de Graaf (17), Medisch Centrum Alkmaar, R. ten Houten (17),

Ziekenhuisgroep Twente, J.C. Baart (15), Haga Ziekenhuis, R.W.M. Keunen

(14), Meander Medisch Centrum, W.G.H. Oerlemans (14), Westfries Gasthuis,

D. Broere (14), Leids Universitair Medisch Centrum, C.S.M. Straathof (13),

Groene-Hart ziekenuis, G.A.M. Verheul (13), Slingeland Ziekenhuis, C.J.W. van

de Vlasakker (13), Universitair Medisch Centrum Groningen, R.H. Enting (13),

OLVG, I.N. van Schaik (12), Tweesteden Ziekenhuis, J.P.L. van der Plas (12),

Gelre Ziekenhuis, H.P. Bienfait (11), Diakonessenhuis Utrecht, M.H. Christiaans

(10), Rijnstate Ziekenhuis, E.M. Hoogerwaard (10), VU Medisch Centrum,

J.C. Reijneveld (10), Beatrix Ziekenhuis, R.B. Alting van Geusau (9), Catharina

Ziekenhuis, J.N. Berendes (9), Erasmus Medisch Centrum, B.C. Jacobs (9), Isala

Klinieken, J.S.P. van den Berg (9), Rijnland Ziekenhuis, R.J.W. Witteveen (9),

Tergooi Ziekenhuizen, M. Stevens, D. Herderschee (9), Boven-IJ Ziekenhuis,

M.A. Struys (8), Gelderse Vallei Ziekenhuis, C. Jansen (8), Orbis Medical Concern,

H.W.M. Anten (8), Sint Elisabeth Ziekenhuis, G.F.J. Brekelmans (8), Sint Jansdal

Ziekenhuis, T.F.M. Fennis (8), StreekZiekenhuis Midden-twente, J.J.W. Prick (8),

Viecuri Ziekenhuis, P.H.M. Pop (8), Sint Lucas Andreas Ziekenhuis, E.J. Wouda

(7), Sint Franciscus Ziekenhuis, C. Bülens (7), Deventer ziekenhuizen, H.J.M.M.

Lohman (6), Flevo Ziekenhuis, J.P. Blankevoort (6), Jeroen Bosch Ziekenhuis,

H.F. Visee (6), Koningin Beatrix Ziekenhuis, R.C.F. Smits (6), Ziekenhuis de

Lievensberg, P.J.I.M. Berntsen (6), Maasstadziekenhuis, R. Saxena (6), Medisch

Spectrum Twente, J.A.G. Geelen (6), Ziekenhuis Bernhoven, P.R. Schiphof (5),

Kennemer Gasthuis, M. Weisfelt (5), Reinier de Graaf Ziekenhuis, W.J.H.M.

Grosveld (5), Scheper Ziekenhuis, E.V. van Zuilen (5), Slotervaart Ziekenhuis,

I.H. Kwa (5), Sint Laurentius Ziekenhuis, P.H.M.F. van Domburg (5), Sint

Jansgasthuis, R.H.J. Medaer (5), Zaans Medisch Centrum, A. Koppenaal (5),

Medisch Centrum Leeuwarden, W. van der Kamp (5), Antonius Ziekenhuis,

R.S. Holscher (4), Bethesda Ziekenhuis, J.P. Schipper (4), Canisius-Wilhelmina

Ziekenhuis, G.W. van Dijk (4), Albert Schweitzer Ziekenhuis, H. Kerkhoff (4),

Medisch Centrum Haaglanden, M.J.B. Taphoorn (4), Dirksland Ziekenhuis, U.W.

Huisman (4), Elkerliek Ziekenhuis, A.J.M.Kok (4), Franciscus Ziekenhuis, A. van

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Spreeken (4), Gemini Ziekenhuis, P. Admiraal (4), Rivierenland Ziekenhuis, P.J. de

Jong (4), Sint Anna Ziekenhuis, H.B.M. van Lieshout (4), Sint Lucas Ziekenhuis,

A.N. Zorgdrager (4), Vlietland Ziekenhuis, C.J. Gijsbers (4), Ziekenhuis Zevenaar,

A.van de Steen (4), Academisch Ziekenhuis Maastricht, Dr. E.P.M. van Raak

(3), Bronovo Ziekenhuis, M. Gerrits (3), Hofpoort Ziekenhuis, E.J. Wieringa (3),

IJsselmeerziekenhuizen, E.M. Leenders (3), Maasziekenhuis, R.M.J.A.Roebroek

(3), Martini Ziekenhuis Groningen, J.W. Snoek (3), Maxima Medisch Centrum,

A.J. Vermeij (3), Mesos Medisch Centrum, P.H. Wessels (3), Oosterschelde

Ziekenhuis, A.M. Boon (3), Refaja Ziekenhuis, L. Vrooland (3), Röpcke-Zweers

Ziekenhuis, J.G.M. Knibbeler (3), Ruwaard van Putten Ziekenhuis, H.W. ter Spill

(3), Spaarne Ziekenhuis, R.J. Meijer (3), Ziekenhuis De Sionsberg, J.P. Krooman

(2), IJsselland Ziekenhuis, J. Heerema (2), Waterland Ziekenhuis, J.G.W. Oonk

(2), Ziekenhuis Amstelland, D.S.M. Molenaar (2), Ziekenhuis Walcheren, J.P.

Koeman (2), Ziekenhuis Zeeuws-Vlaanderen, W. Hoefnagels (2), Ziekenhuis de

Tjongerschans, R.F. Duyff (2), Ziekenhuis Delfzicht, J.A. Don (1), Diaconessenhuis

Meppel, E.J.V. Keuter (1), Havenziekenhuis, R.J.W. Dunnewold (1), Ziekenhuis

Nij Smellinghe, K.D. Beintema (1), Rode Kruis Ziekenhuis, L. Zegerius (1), Sint

Antonius Ziekenhuis, H.W. Mauser (1), Wilhelmina Ziekenhuis, A.E. Bollen (1).

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References

1. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-59.


3. Tauber MG, Khayam-Bashi H, Sande MA. Effects of ampicillin and corticosteroids on brain water content, cerebrospinal fluid pressure, and cerebrospinal fluid lactate levels in experimental pneumococcal meningitis. J Infect Dis 1985;151:528-34.

4. Scheld WM, Dacey RG, Winn HR, Welsh JE, Jane JA, Sande MA. Cerebrospinal fluid outflow resistance in rabbits with experimental meningitis. Alterations with penicillin and methylprednisolone. J Clin Invest 1980;66:243-53.

5. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-56.

6. Scarborough M, Gordon SB, Whitty CJ et al. Corticosteroids for bacterial meningitis in adults in sub-Saharan Africa. N Engl J Med 2007;357:2441-50.

7. Nguyen TH, Tran TH, Thwaites G et al. Dexamethasone in Vietnamese adolescents and adults with bacterial meningitis. N Engl J Med 2007;357:2431-40.

8. Peltola H, Roine I, Fernandez J, et al. Adjuvant glycerol and-or dexamethasone to improve the outcomes of childhood bacterial meningitis: a prospective, randomized double-blind, placebo-controlled trial. Clin Infect Dis 2007;45:1277-1286

9. Molyneux EM, Tembo M, Kayira K et al. The effect of HIV infection on paediatric bacterial meningitis in Blantyre, Malawi. Arch Dis Child 2003;88:1112-8.

10. van de Beek D, Farrar JJ, de Gans J, et al. Adjunctive dexamethasone in bacterial meningitis: a meta-analysis of individual patient data. Lancet Neurol 2010;9:254-263.

11. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53.

12. Tunkel AR, Hartman BJ, Kaplan SL et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39:1267-84.

13. Chaudhuri A, Martinez-Martin P, Kennedy PG et al. EFNS guideline on the management of community-acquired bacterial meningitis: report of an EFNS Task Force on acute bacterial meningitis in older children and adults. Eur J Neurol 2008;15:649-59.

14. Statistics Netherlands. Statline, Voorburg/Heerlen, 2001. 2009;Available at: URL: www.cbs.nl.

15. Jennett B, Teasdale G. Management of head injuries. 2 ed. Philadelphia: F.A.Davis; 1981.

16. Spanos A, Harrell FE, Jr., Durack DT. Differential diagnosis of acute meningitis. An analysis of the predictive value of initial observations. JAMA 1989;262:2700-7.

17. van de Beek D, de Gans J, Spanjaard L, Vermeulen M, Dankert J. Antibiotic guidelines and antibiotic use in adult bacterial meningitis in The Netherlands. J Antimicrob Chemother 2002;49:661-6.

18. Benson K, Hartz AJ. A comparison of observational studies and randomized, controlled trials. N Engl J Med 2000;342:1878-86.

19. MacLehose RR, Reeves BC, Harvey IM, Sheldon TA, Russell IT, Black AM. A systematic review of comparisons of effect sizes derived from randomised and non-randomised studies. Health Technol Assess 2000;4:1-154.

20. Miettinen OS. The need for randomization in the study of intended effects. Stat Med 1983;2:267-71.

21. Vandenbroucke JP. Observational research, randomised trials, and two views of medical science. PLoS Med 2008;5:e67.

22. Opal SM, Girard TD, Ely EW. The immunopathogenesis of sepsis in elderly patients. Clin Infect Dis 2005;41(suppl 7):S504-S512.

23. Mai NTH, Hoa NT, Nga TVT, et al. Streptococcus suis meningitis in adults in Vietnam. Clin Infect Dis 2008;46:659-667.

24. Swartz MN. Bacterial meningitis--a view of the past 90 years. N Engl J Med 2004;351:1826-8.

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Chapter 6

Hearing loss in adults surviving pneumococcal meningitis

is associated with otitis and pneumococcal serotype


Matthijs C. Brouwer

Arie van der Ende

Erik F. Hensen


Clinical Microbiology and Infection, 2012;18(9):849-55.

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Abstract

We assessed the incidence of hearing loss and relation with clinical

characteristics and pneumococcal serotypes in adults surviving pneumococcal

meningitis.

We analyzed hearing loss in 531 adults surviving pneumococcal meningitis

included in two prospective nationwide cohort studies performed from April

1998 through October 2002 and March 2006 through January 2009. Hearing

loss was evaluated on admission and discharge for all patients. Severe hearing

loss was assessed by pure tone average on audiology and corrected for age, or

by the combination of hearing loss on discharge and a score on the Glasgow

Outcome Scale below 5, which could not be explained by other neurological

sequelae.

A total of 531 episodes of pneumococcal meningitis with non-lethal outcome

were included. Predisposing conditions for pneumococcal meningitis were

present in the majority of patients (64%), most commonly otitis (36%). Hearing

loss was present at discharge in 116 episodes (22%) and was classified as mild

in 53% and severe in 47%. Hearing loss was related with otitis (odds ratio [OR]

2.58; 95% confidence interval [CI] 1.66-4.02; p<0.001) and inversely related

with serotype 23F infection (OR 0.36; 95% CI 0.13-0.98; p=0.025), but not with

parameters of disease severity or indicators of cerebrospinal fluid inflammation

severity. Meningitis due to pneumococcal serotype 3 was associated with the

highest rate of hearing loss.

We conclude that hearing loss frequently complicates pneumococcal

meningitis. Otitis was a risk factor for hearing loss, but not disease severity.

Hearing loss was inversely related with serotype 23F infection. Otitis and

resulting perilympathic inflammation contribute to meningitis-associated

hearing loss.

Background

Bacterial meningitis is a severe and life-threatening infectious disease.1

Streptococcus pneumoniae is the most severe cause of bacterial meningitis

and currently accounts for ~70% of all cases of community-acquired bacterial

meningitis.2 Hearing loss commonly complicates the clinical course of

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pneumococcal meningitis and is an important cause of disability, with reported

rates of 7% to 36% among survivors of pneumococcal meningitis. 1, 3-6

Few studies have identified clinical risk factors for meningitis-associated

hearing loss.3-12 The most commonly described risk factor for hearing loss

were age, a low level of consciousness, infection with S. pneumoniae, and

cerebrospinal fluid (CSF) and serum parameters of inflammation.3-12 A recent

retrospective cohort study showed that advanced age, female sex, presence of

co-morbidity, and pneumococcal serotype were associated with an increased

risk of hearing loss. 4 The influence of serotype on hearing loss had not been

described previously and may influence vaccination policy and development.

In the current study we assessed the incidence of hearing loss in two

prospective nationwide studies on pneumococcal meningitis and studied the

role of pneumococcal serotypes in the development of hearing loss.

Methods

We pooled data from patients with pneumococcal meningitis from two

prospective nationwide cohort studies with similar design.3, 13 In these studies

adults (defined as patients older than 16 years of age) were included who had

bacterial meningitis defined by positive CSF culture, and were listed in the

database of the Netherlands Reference Laboratory for Bacterial Meningitis

from April 1998 through October 2002, and from March 2006 to January 2009.

This laboratory receives CSF isolates from approximately 85% of all patients

with bacterial meningitis in the Netherlands (population, 16.2 million). 3,13 Daily

updates were provided of hospitals where patients with bacterial meningitis

had been admitted in the preceding 2 to 6 days. Physicians were informed about

the study by telephone. Patients or their legal representatives received written

information concerning the study and were asked to give written informed

consent for participation. Case record forms were used to collect data. Patients

with negative CSF cultures or hospital-associated meningitis were excluded.

Patients with an altered immune status due to the use of immunosuppressive

drugs or splenectomy, diabetes mellitus, or alcoholism were considered

immunocompromised, as were patients infected with HIV.

Outcome was graded according to the Glasgow Outcome Scale. A score of 1 on

this scale indicates death; a score of 2 a vegetative state (the patient is unable to

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interact with the environment); a score of 3 severe disability (the patient is unable

to live independently but can follow commands); a score of 4 moderate disability

(the patient is capable of living independently but unable to return to work or

school); and a score of 5 mild or no disability (the patient is able to return to work

or school). A favorable outcome was defined as a score of 5, and an unfavorable

outcome as a score of 1 to 4. The Glasgow Outcome Scale is a well-validated

instrument with good interobserver agreement.14 At discharge, all surviving

patients underwent a neurologic examination performed by a neurologist.

Hearing loss was evaluated on admission, during admission and on discharge

for all patients. Audiological follow-up was performed at the discretion of

the treating physician. Audiograms performed within one year of admission

were retrieved in patients with a history of hearing loss on admission,

during admission or at discharge. Audiograms were re-evaluated by an

otolaryngologist (EH) and a neurologist (SH). Pure tone audiometry at 500,

1000, 2000 and 4000 Hz was used to calculate the pure tone average (PTA). The

PTA was corrected for any co-existent conductive hearing loss by subtracting

the air-bone gap. Finally, an age- and sex-matched dataset was used to correct

the PTA values.15 By correcting for conductive hearing loss, age and sex, we

obtained the corrected pure tone average (cPTA). We categorized patients in

four categories: patients with a cPTA of <10 decibels (dB) uni- or bilaterally (no

hearing loss), patients with a cPTA of 10-30 dB uni- or bilaterally (mild hearing

loss), patients with a cPTA of 31-70 dB uni- or bilaterally (moderate hearing loss)

and patients with a cPTA >70dB (severe hearing loss).4 We analyzed patients

with any hearing loss and those with moderate to severe hearing loss, which

was defined as by cPTA>30. Patients with the combination of hearing loss

at discharge and a score on the Glasgow Outcome Scale below 5, which was

not caused by other neurological sequelae, were also categorized as having

moderate to severe hearing loss. Clinical hearing loss in the case record form

was not specified as uni- or bilateral hearing loss.

The Mann-Whitney U test was used to identify differences between groups with

respect to continuous variables, and dichotomous variables were compared by

use of the chi2 test. All statistical tests were 2-tailed, and a p value of <0.05 was

regarded as significant. Analyses were undertaken with PASW software, version

18 (SPSS, Armonk, NY, USA). We used logistic regression analysis to assess the

association between potential prognostic factors and the probability of any or

severe hearing loss.

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Results

A total of 709 episodes of pneumococcal meningitis in 697 patients were

included in the two cohort studies; 352 in 1998-2002 and 357 in 2006-2009. 178

episodes were fatal (25%), so 531 episodes of adult pneumococcal meningitis

were available for evaluation in this study.

Predisposing conditions for pneumococcal meningitis were present in 342

patients (64%), and consisted of otitis in 195 patients (36%; Table 1). On

presentation classic symptoms and signs of bacterial meningitis were present

in a large proportion of patients. Headache occurred in 87% of episodes, neck

stiffness in 81%, fever in 86%, and a change in mental status (defined by a

Glasgow Coma Scale score below 14) in 81%. In 15% of episodes, the patients

were comatose on admission, and in 9% of episodes, a hemiparesis was

present on presentation. A lumbar puncture was performed in all patients, and

the median white blood cell (WBC) count was 3004 (interquartile range [IQR],

893-8121).

Hearing loss was noted in 187 of 531 episodes (35%); on or during admission

only in 71 patients, and at discharge in 116 patients (Table 2). Audiological

examination was performed in 82 of 187 episodes with hearing loss (44%).

According to our predefined criteria, hearing loss was present in 73 audiograms

and was classified as mild in 30 episodes (37%), moderate in 25 (30%) and

severe in 18 episodes (22%). In the other 105 episodes complicated by hearing

loss in which audiology was not performed, hearing loss was still present on

discharge in 43 (41%) of these patients. In 12 (28%) of these episodes without

audiograms hearing loss was classified as severe hearing loss as it caused

unfavorable outcome. Combining the clinical and audiology data we concluded

that any hearing loss was present on discharge in 116 episodes (22%). The

degree of hearing loss was classified as moderate to severe hearing loss in 55

episodes (47%). Clinical features in patient with audiological examination and

with clinical hearing loss were similar, except for diastolic hypotension, which

was more common on presentation in patients receiving audiological follow-

up (17% vs. 2%, p=0.03). Hearing loss in audiograms was most pronounced in

the highest frequency (4000Hz).

In a univariate analysis, the presence of otitis on presentation was associated

with hearing loss at discharge (odds ratio [OR] 2.58; 95% confidence interval [CI]

1.66-4.02; p< 0.001; Table 2). Of the 195 patients with otitis, 64 developed hearing

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loss (33%). The association with otitis was present in patients with audiologically

confirmed hearing loss (p<0.001) and in patients with clinical hearing loss

(p=0.003). Clinical severity, as reflected in scores on the Glasgow Coma Scale,

and parameters of CSF inflammation, were not predictive for the development

of hearing loss. Patients with severe hearing loss had lower CSF/blood glucose

ratios, as compared with those without severe hearing loss (ratio of 0.03 [IQR,

0.00-0.08] vs. 0.05 [IQR 0.01-0.25]; p=0 .02), but all other clinical and laboratory

Table 1. Baseline characteristics in 531 episodes of pneumococcal meningitis

Characteristic Value

Age (years) 59 (45-68)

Male sex 242/531 (46%)

Duration of symptoms >24 h 251/509 (49%)

Predisposing conditions 342/531 (64%)

Otitis 195/522 (37%)

Sinusitis 90/509 (18%)

Pneumonia 66/523 (13%)

Immunocompromise 111/530 (21%)

Symptoms and signs on admission

Headache 417/479 (87%)

Neck stiffness 420/519 (81%)

Fever 385/447 (86%)

Diastolic blood pressure <60 mmHg 39/523 (7%)

GCS on admission 11 (9-13)

<14 (indicating change in mental status) 428/531 (81%)

<8 (indicating coma) 80/531 (15%)

Focal neurologic abnormalities on admission

Hemiparesis 47/498 (9%)

Cranial nerve palsy 66/498 (13%)

Hearing loss 26/454 (6%)

Indices of CSF inflammation

White cell count (cells/mm3) 3004 (893-8121)

CSF protein (g/L) 3.9 (2.4-5.9)

CSF/Blood glucose ratio 0.04 (0.01-0.24)

CSF Pressure (cm H2O) 40 (29-50)

Blood tests

ESR, mm/hr 42 (22-72)

C-reactive protein, mg/L 185 (92-203)

Thrombocyte count, platelets/mm3 207 (163-262)

Numbers are number/number evaluated or median (interquartile range [IQR]). GCS: Glasgow coma scale. CSF: cerebrospinal fluid. ESR: erythrocyte sedimentation rate. CSF white cell count was available for 498 episodes. CSF protein was available for 492 episodes. CSF/Blood glucose ratio was available for 486 episodes. CSF pressure was available for 172 episodes. ESR was available for 369 episodes. C-reactive protein was available for 389 episodes. Thrombocyte count was available for 501 episodes.

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Table 2. Characteristics in 531 episodes of pneumococcal meningitis with and without hearing loss.

Characteristic Any hearing loss No hearing loss p-value

No. of episodes 116 415

Age, y 56±14 57±15 0.49

Male sex 49/116 (42%) 193/415 (47%) 0.41

Duration of symptoms >24 h 53/111 (48%) 198/398 (50%) 0.71


Otitis 64/116 (55%) 131/406 (32%) <0.001

Sinusitis 24/109 (22%) 66/400 (17%) 0.18

Pneumonia 15/114 (13%) 51/409 (12%) 0.84

Immunocompromise 22/116 (19%) 89/414 (21%) 0.55

Symptoms and signs on admission

Headache 96/108 (89%) 321/371 (87%) 0.52

Neck stiffness 91/114 (80%) 329/405 (81%) 0.73

Diastolic blood pressure <60 mmHg 13/115 (11%) 26/408 (6%) 0.08

Hemiparesis 12/104 (12%) 35/394 (9%) 0.41

Score on GCSb

<14 (change in mental status) 92/115 (80%) 336/415 (81%) 0.81

<8 (coma) 13/115 (11%) 67/415 (16%) 0.20

Indices of CSF inflammation

White cell count (cells/mm3)a 3370 (762-8692) 2933 (902-8000) 0.40

CSF protein (g/L)b 4.1 (2.8-5.8) 3.8 (2.3-5.9) 0.27

CSF/Blood glucose ratioc 0.04 (0.00-0.21) 0.05 (0.01-0.24) 0.57

Numbers are number / number evaluated or median (IQR). GCS: Glasgow coma scale. CSF: cerebrospinal fluid. a CSF white cell count was available for 498 episodes. b CSF protein was available for 492 episodes. c CSF/Blood glucose ratio was available for 486 episodes.

characteristics were similar between groups. In a multivariate analysis with

possible predictors of hearing loss (sinusitis, otitis, diastolic hypotension and

coma on admission), otitis on admission remained the only predictor of hearing

loss (p<0.001). Treatment data for otitis were not available for a sufficient

number of patients to asses the influence of, for example, mastoidectomy on

hearing loss on discharge.

Serotype analysis was performed for 504 of 531 pneumococcal isolates (95%;

Table 3). Most common capsular serotypes were 3, 7F, 23F, 14, 6B, 19F, together

accounting for 47% of isolates. The coverage of the 7-, 10-, and 13-valent

vaccine of these isolates would be 40%, 53%, and 68%, respectively. Compared

to the reference serotype 3 (the most common serotype) the risk of hearing

loss was lower for all other serotypes. Any hearing loss during clinical course

occurred less frequently in patients infected with S. pneumoniae serotype 14

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(OR 0.28; 95% CI 0.09-0.80; p=0.007) and serotype 23F (OR 0.36; 95% CI 0.13-

0.98; p=0.025), as compared with serotype 3. Infection with the S. pneumoniae

serotype 23F was also associated with less hearing loss at discharge (OR 0.32;

95%CI 0.08-1.19; p=0.05) and showed a trend towards less severe hearing loss

at discharge (OR 0.19; 95% CI 0.01-1.71; p=0.10), but confidence intervals were

wide because of small numbers. S. pneumoniae serotype 14 was not related

with hearing loss at discharge.

Table 3. Distribution of pneumococcal serotypes in 504 episodes of pneumococcal meningitis with and without hearing loss

Serotype Frequency(%)

Hearing loss during clinical courseNo. (%)

Odds ratio (95% confidence interval)

p Hearing loss at dischargeNo. (%)

Odds ratio (95% confidence interval)

p

14a 38 (8) 8 (30) 0.28 (0.09–0.80) 0.007 6 (16) 0.52 (0.15-1.70) 0.23

19Fa 28 (6) 10 (36) 0.58 (0.20–1.66) 0.26 8 (29) 1.11 (0.35-3.51) 0.85

22F 23 (5) 7 (30) 0.46 (0.14–1.46) 0.14 5 (22) 0.77 (0.20-2.83) 0.66

23Fa 39 (8) 10 (26) 0.36 (0.13–0.98) 0.025 4 (10) 0.32 (0.08-1.19) 0.05

3 49 (10) 24 (49) 1.00 (reference) - 13 (27) 1.00 (reference) -

4a 22 (4) 9 (41) 0.72 (0.23–2.24) 0.53 6 (27) 1.04 (0.29-3.67) 0.94

6Ba 33 (7) 12 (36) 0.60 (0.22–1.61) 0.26 8 (24) 0.89 (0.28-2.74) 0.82

7F 48 (10) 18 (38) 0.63 (0.26–1.52) 0.25 9 (19) 0.64 (0.22-1.85) 0.84

8 31 (6) 12 (39) 0.66 (0.24–1.81) 0.37 8 (26) 0.96 (0.31-3.00) 0.94

9Va 27 (5) 11 (41) 0.72 (0.25–0.52) 0.49 8 (30) 1.17 (0.36-3.72) 0.77

Other serotypes

166 (33) 58 (35) - - 38 (22) - -

Total 504 (100) 179 (36) - - 113 (22) - -a Included in 7 valent pneumococcal conjugate vaccine.

Initial antibiotic treatment consisted of penicillin or amoxicillin monotherapy in

41% of patients, third-generation cephalosporin monotherapy in 22% patients,

a combination of penicillin or amoxicillin and a third-generation cephalosporin

in 27% patients, and different regimens were used in 10% of patients. Use of

aminoglycosides was reported in 25 episodes and hearing loss at discharge

was found in 3 of those patients (12%). Aminoglycosides are associated with

ototoxicity and hearing loss, however, there was no increased risk of hearing

loss in patients receiving aminoglycosides in our study.16

Between the first and second cohort study, after the publication of a clinical

trial and meta-analysis, 17,18 adjunctive dexamethasone was introduced as

routine treatment in the Netherlands.13 In our patient cohorts, the standard

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regimen of dexamethasone, 10 mg four times daily, started with of before the

first dose of antibiotics, was administered in 240 of 531 episodes (45%). The

proportion of patients with hearing loss treated with or without the standard

regimen dexamethasone was similar (48 of 240 [20%] vs. 68 of 291 [23%];

p=0.35). The proportion of patients with severe hearing loss treated with or

without standard regimen dexamethasone was also similar (20 of 240 [8.3%]

vs. 35 of 291 [12%]; p=0.16).

Discussion

The current study shows that hearing loss frequently complicates pneumococcal

meningitis. In our adult population, hearing loss was present in about one in four

patients surviving their disease. Previous studies have reported rates of hearing

loss varying from 7 to 36%. A recent retrospective Danish study based on a

nationwide registration during a 5-year period (1999-2003) reported a rate of

54%. The majority of these patients had hearing loss on audiological examination

only, without complaints. Our study was performed nationwide and, therefore,

we were able to study a representative sample of adults surviving pneumococcal

meningitis. Our prospective approach allowed us to collect comprehensive data

on signs and symptoms, clinical course and outcome.

We identified otitis as the main risk factor for hearing loss (OR 2.58 CI 1.66-4.02;

p<0.001). This is consistent with findings of a previous prospective cohort study

that reported a similar rate of hearing loss among patients with pneumococcal

meningitis and otitis (33%).7 Other studies have found a relation between markers

of CSF inflammation and hearing loss.4 Our finding that hearing loss is related

with otitis, but not with disease severity or levels of CSF inflammation, suggests

a role for otitis and resulting perilympathic inflammation in the pathogenesis

of meningitis-associated hearing loss, consistent with the aggravated high-

frequency loss in our patients. Research in animals and patients with bacterial

meningitis showed that the main site of the lesion in meningitis-associated

hearing is the cochlea.5 Predominant morphological correlates of acute

meningitis-associated hearing loss are damage to the blood-labyrinth barrier, hair

cells and spiral ganglion.5 However, the route of entry of bacteria into the cochlea

has been subject to debate. Bacteria can reach the cochlea via the bloodstream,

the vestibulocochlear nerve, or through the perilymphatic duct. Klein et al5

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concluded that the most probable route of infection is a spread of bacteria from

the subarachnoid space through the perilymphatic duct to the perilymphatic

space of the cochlea. However, in otitis media, toxic substances (bacterial

products and inflammatory mediators) may pass from the middle ear passage

through the round window membrane, causing perilympathic inflammation,

starting within the cochlear basal turn. 19 This localization is consistent with the

prevalence of sensorineural hearing loss at higher frequencies as found in patients

with post-meningitis hearing loss. 5 Subsequently, these toxic substances may

cause permanent cochlear damage resulting in hearing loss. 19 The association

of hearing loss with otitis instead of severity of CSF inflammation would further

be supported if the side of otitis corresponded with the side of hearing loss. The

side of otitis, mastoiditis and hearing loss was not systematically registered in our

study, which is a limitation of our results and leaves room for both versions of the

pathophysiology of hearing loss in pneumococcal meningitis.

Adjunctive dexamethasone therapy was not associated with a lower risk

on hearing loss among survivors of pneumococcal meningitis, although

a trend was found for reduced risk of disabling hearing loss in patients

receiving dexamethasone. Adjunctive anti-inflammatory therapies have

been investigated to decrease rates of post-meningitis hearing loss. Systemic

adjunctive steroid treatment reduced long-term hearing loss in experimental

pneumococcal meningitis. 20 Clinical trials and recent meta-analyses have shown

that adjunctive dexamethasone therapy prevents hearing loss in patients with

bacterial meningitis. 21, 22 We recently showed that dexamethasone therapy

has been implemented on a large scale as adjunctive treatment of adults with

pneumococcal meningitis in the Netherlands. 13 The prognosis of pneumococcal

meningitis on a national level has substantially improved after the introduction

of adjunctive dexamethasone therapy. 13 However, as dexamethasone improves

outcome and reduces mortality, a larger proportion of patients survive and are

at risk for developing hearing loss. Other adjunctive therapies to prevent hearing

loss, i.e., antioxidant therapy and neurotrophin-3, have been investigated in

animal models and may present a promising future treatment options. 23, 24

Our study has several limitations. As audiological examination was performed on

the discretion of the treating physician, only 16% of patients received audiological

examination. We extended the definition of hearing loss to describe all patients

with clinical hearing loss on discharge, even though bedside assessment of hearing

loss is of limited value. 4 The limited availability of audiological examinations

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hinders the interpretation of our results, but reflects clinical practice. Furthermore,

in patients with poor clinical outcome, hearing loss may be masked by clinical

condition, e.g. decreased level of consciousness. Therefore these patients may

have been missed. Finally treatment data for otitis (mastoidectomy) were not

reported, which is be a possible confounder for hearing loss on discharge.

However, this does not affect the identified association between otitis and hearing

loss as a possible treatment effect is included in this analysis.

Our reference pneumococcal serotype, serotype 3, was associated with the

highest risk on hearing loss. Serotype 23F was associated with a significant lower

risk of hearing loss, as compared with the serotype 3. The Danish study showed

fewer hearing loss in patients infected with serotype 6B, but also found the

highest risk in patients infected with serotype 3. 4 A systematic review and meta-

analysis of serotype-specific disease outcomes for patients with pneumonia

and meningitis showed that the relative risk of death in patients infected

with serotype 3 was increased. 25 Experimental animal studies are needed to

further elucidate the role of different pneumocococcal serotypes on the risk for

meningitis-associated hearing loss.

The high incidence of hearing loss in patients with pneumococcal meningitis

warrants consultation of an otolaryngologist in patients with suspected hearing

loss. 1 Otitis on presentation is common in pneumococcal meningitis and may

require invasive treatment to remove the focus of infection. Hearing loss may not

be clinically evident in patients (e.g. those with an altered state of consciousness

or patients requiring mechanical ventilation). The threshold for perfoming

audiometric evaluation should therefore be low. In patients with hearing loss due

to pneumococcal meningitis, obliteration of the cochlear lumen may occur in the

weeks after the hearing loss developed. This will further diminish sensorineural

hearing and adversely affects hearing revalidation options such as cochlear

implantation, even in patients with initial mild hearing loss. 28, 29 In these cases,

cochlear implantation before obliteration has occurred may prevent permanent

disabling hearing impairment. 26, 27 Therefore, otolaryngological evaluation in

these patients should be performed in the acute phase of disease.

Acknowledgements

We thank Floortje Ruijter for her assistance with retrieving audiograms.

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References

1. van de Beek D, de Gans J, Tunkel AR, Wijdicks EFM. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:46-55.

2. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 2010;23:467-92.

3. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-59.

4. Worsoe L, Caye-Thomasen P, Brandt CT, Thomsen J, Ostergaard C. Factors associated with the occurrence of hearing loss after pneumococcal meningitis. Clin Infect Dis 2010;51:917-24.

5. Klein M, Koedel U, Kastenbauer S, Pfister HW. Nitrogen and oxygen molecules in meningitis-associated labyrinthitis and hearing impairment. Infection 2008;36:2-14.

6. Dodge PR, Davis H, Feigin RD, Holmes SJ, Kaplan SL, Jubelirer DP, Stechenberg BW, Hirsh SK. Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis. N Engl J Med 1984;311:869-74.

7. Ostergaard C, Konradsen HB, Samuelsson S. Clinical presentation and prognostic factors of Streptococcus pneumoniae meningitis according to the focus of infection. BMC Infect Dis 2005;5:93.


9. Weisfelt M, de Gans J, van der Poll T, van de Beek D. Pneumococcal meningitis in adults: new approaches to management and prevention. Lancet Neurol 2006;5:332-42.

10. Peltola H, Roine I, Fernández J, González Mata A, Zavala I, Gonzalez Ayala S, et al. Hearing impairment in childhood bacterial meningitis is little relieved by dexamethasone or glycerol. Pediatrics 2010;125:e1-8.

11. Adachi N, Ito K, Sakata H. Risk factors for hearing loss after pediatric meningitis in Japan. Ann Otol Rhinol Laryngol 2010;119:294-6.

12. Jit M. The risk of sequelae due to pneumococcal meningitis in high-income countries: a systematic review and meta-analysis. J Infect 2010;61:114-24.

13. Brouwer MC, Heckenberg SG, de Gans J, Spanjaard L, Reitsma JB, van de Beek D. Nationwide implementation of adjunctive dexamethasone therapy for pneumococcal meningitis. Neurology 2010;75:1533-9.

14. Jennett B, Teasdale G. Management of Head Injuries, 2ed. Philadelphia: FA Davis; 1981.

15. ISO institute. Acoustics -- Statistical distribution of hearing thresholds as a function of age. Edition 2, Stage: 90.92,ICS:13.140,2000.

16. Dobie RA, Black FO, Pezsnecker SC, Stallings VL. Hearing loss in patients with vestibulotoxic reactions to gentamicin therapy. Arch Otolaryngol Head Neck Surg 2006;132:253-7.

17. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-56

18. van de Beek D, de Gans J, McIntyre P, Prasad K. Steroids in adults with acute bacterial meningitis: a systematic review. Lancet Infect Dis 2004;4:139-43.

19. Joglekar S, Morita N, Cureoglu S, Schachern PA, Deroee AF, Tsuprun V, et al. Cochlear pathology in human temporal bones with otitis media. Acta Otolaryngol 2010;130:472-6.

20. Worsøe L, Brandt CT, Lund SP, Østergaard C, Thomsen J, Cayé-Thomasen P. Systemic steroid reduces long-term hearing loss in experimental pneumococcal meningitis. Laryngoscope 2010;120:1872-9.

21. van de Beek D, Farrar JJ, de Gans J, Mai NT, Molyneux EM, Peltola H, et al. Adjunctive dexamethasone in bacterial meningitis: a meta-analysis of individual patient data. Lancet Neurol 2010;9:254-63.


23. Klein M, Koedel U, Pfister HW, Kastenbauer S. Meningitis-associated hearing loss: protection by adjunctive antioxidant therapy. Ann Neurol 2003;54:451-8.

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24. Demel C, Hoegen T, Giese A, Angele B, Pfister HW, Koedel U, Klein M. Reduced spiral ganglion neuronal loss by adjunctive neurotrophin-3 in experimental pneumococcal meningitis. J Neuroinflammation 2011;8:7.

25. Weinberger DM, Harboe ZB, Sanders EA, Ndiritu M, Klugman KP, et al. Association of serotype with risk of death due to pneumococcal pneumonia: a meta-analysis. Clin Infect Dis 2010;51:692-9.

26. Cohen NL, Hirsch BE. Current status of bacterial meningitis after cochlear implantation. Otol Neurotol 2010;31:1325-8.

27. Merkus P, Free RH, Mylanus EA, Stokroos R, Metselaar M, van Spronsen E, Grolman W, Frijns JH; 4th Consensus in Auditory Implants Meeting. Dutch Cochlear Implant Group (CI-ON) consensus protocol on postmeningitis hearing evaluation and treatment. Otol Neurotol 2010;31:1281-6.

28. Nabili V, Brodie HA, Neverov NI, Tinling SP. Chronology of labyrinthitis ossificans induced by Streptococcus pneumoniae meningitis. Laryngoscope 1999;109:931-5.

29. Waltzman SB, Fisher SG, Niparko JK, Cohen NL. Predictors of postoperative performance with cochlear implants. Ann Otol Rhinol Laryngol Suppl 1995;165:15-8.

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Chapter 7

Complement component 5 contributes to poor disease

oucome in humans and mice with pneumococcal meningitis

Bianca Woehrl*


Carmen Murr


Frank Baas

Hans W. Pfister

Aeilko H. Zwinderman

B. Paul Morgan

Scott R. Barnum

Arie van der Ende

Uwe Koedel‡

Diederik van de Beek‡

*, ‡: both authors contributed equally

Journal of Clinical Investigation, 2011;121(10):3943-3953

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Abstract

Pneumococcal meningitis is the most common and severe form of bacterial

meningitis. Fatality rates are substantial, and long-term sequelae develop in

about half of survivors. Disease outcome has been related to the severity of

the proinflammatory response in the subarachnoid space. The complement

system, which mediates key inflammatory processes, has been implicated

as a modulator of pneumococcal meningitis disease severity in animal

studies. Additionally, SNPs in genes encoding complement pathway proteins

have been linked to susceptibility to pneumococcal infection, although no

associations with disease severity or outcome have been established. Here, we

have performed a robust prospective nationwide genetic association study in

patients with bacterial meningitis and found that a common nonsynonymous

complement component 5 (C5) SNP (rs17611) is associated with unfavorable

disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients

with bacterial meningitis correlated with several clinical indicators of poor

prognosis. Consistent with these human data, C5a receptor–deficient mice

with pneumococcal meningitis had lower CSF wbc counts and decreased

brain damage compared with WT mice. Adjuvant treatment with C5-specific

monoclonal antibodies prevented death in all mice with pneumococcal

meningitis. Thus, our results suggest C5-specific monoclonal antibodies could

be a promising new antiinflammatory adjuvant therapy for pneumococcal

meningitis.

Introduction

Community-acquired bacterial meningitis continues to exact a heavy toll, even

in developed countries, despite the implementation of childhood vaccination

programs and effective antimicrobial agents.1, 2 The most common etiologic

agents of bacterial meningitis are Streptococcus pneumoniae and Neisseria

meningitidis, with the first bacterium responsible for two-thirds of cases in

Europe and the United States.1 The fatality rates in patients with meningitis

caused by these microorganisms are substantial, at 26% and 9%, respectively,

and long-term sequelae, including hearing loss, focal neurological deficit, and

cognitive impairment, develop in about half of survivors. 1, 3

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Experimental animal models have shown that outcome in bacterial meningitis

is related to the severity of inflammation in the subarachnoid space, and it was

suggested that outcome could be improved by modulation of the inflammatory

response, for example, with dexamethasone.4 Many randomized clinical trials of

dexamethasone in bacterial meningitis have been performed, but results have

remained ambiguous.5-8 An individual patient data meta-analysis of 5 large

recent trials showed no effect of dexamethasone.7 A prospective cohort study

showed a decrease in mortality from 30% to 20% in adults with pneumococcal

meningitis after nationwide implementation of dexamethasone in the

Netherlands.9 New adjunctive therapies are needed to improve the prognosis

of bacterial meningitis.

Genetic association studies may reveal new targets for adjuvant therapies.10

Genetic defects in the complement system have been studied in patients

with extreme phenotypes of meningitis, particularly those with familial or

recurrent disease, focusing on susceptibility to invasive pneumococcal and

meningococcal disease.11 The complement system can be divided into 3

activation pathways (the classical, lectin, and alternative pathways), which all

converge on a common terminal pathway.12 An essential step in the classical and

lectin pathways is cleavage of complement component C2 into its fragments,

C2a and C2b. A retrospective study, including 40,000 patients with suspected

complement deficiency, identified 40 individuals with C2 deficiency due to a 28-

bp deletion.13 A history of invasive infections, mainly pneumococcal infections,

was found in 23 (58%) of these individuals.13 The formation of the alternative

pathway C3 convertase complex (C3bBb) is a crucial step in the alternative

pathway and requires complement factor D (fD).12 fD deficiency due to

uncommon SNPs has been described in cases and families with meningococcal

and pneumococcal infections.14, 15 C3bBb is stabilized by properdin, and

properdin deficiency predisposes to meningococcal disease due to serogroups

W135 and Y; one-third of patients with meningococcal disease caused by these

serotypes are properdin deficient. 16 The common terminal pathway consists

of complement components C5–C9, and activation forms the anaphylatoxin

C5a, a strong proinflammatory mediator, and the membrane attack complex

(MAC), which creates pores in the bacterial cell wall.12 Deficiencies in these late

complement components have been recognized as a cause of recurrent and

familial meningococcal infections.

Case-control studies subsequently assessed the effect of SNPs in complement

24407 Heckenberg.indd 111 25-02-13 11:11

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112

genes on susceptibility to pneumococcal and meningococcal disease in

the general population.11 A meta-analysis of studies on 3 SNPs in mannose-

binding lectin showed an association of homozygosity for variant alleles with

pneumococcal invasive disease (odds ratio [OR], 2.58; 95% CI, 1.38–4.80).11

This soluble pattern recognition molecule activates the lectin pathway upon

binding to microorganisms.12 Factor H (fH) regulates the alternative pathway by

inactivating C3bBb (12). The fH –496C/C genotype was found to be associated

with meningococcal disease (OR, 2.0; 95% CI, 1.3–3.2).17 Most of the candidate

gene approach studies lacked power to detect true associations.11 Recently, a

genomewide association study (GWAS) on host susceptibility to meningococcal

disease identified a locus in the complement factor H (CFH) region, providing

the first convincing evidence for a role of SNPs in complement genes in

susceptibility to infections.18 Little is known about the role of complement

SNPs in bacterial meningitis, and so far no associations with disease severity

or outcome have been reported in case-control studies for complement SNPs

or GWAS. 11

Studies in animal models have provided evidence for involvement of the

complement system in modulating severity of pneumococcalmeningitis. In

rabbits depleted of C3 by administering cobra venom factor, intracisternal

inoculation of S. pneumoniae resulted in higher bacterial titers in the

cerebrospinal fluid (CSF) than in complement-sufficient control animals. 19 Other

studies showed an increased pneumococcal outgrowth in the brain and blood

in gene-targeted mice lacking C1q, affecting only the classical pathway; C3,

affecting all complement activation pathways; or the receptor for the opsonin

C3b/iC3b (CR3). 20, 21 C3 deficiency led to diminished brain inflammation,

paralleled by an attenuation of intracranial complications. However, the lack

of CR3-mediated opsonophagocytosis resulted in ineased bacteremia that

worsened outcome. These data provide evidence that the complement system

is important in bacterial meningitis and that antagonizing the detrimental

proinflammatory effects of the complement system without inhibiting its

antimicrobial activity might be a promising adjuvant therapy option.

We performed a prospective nationwide genetic association study in patients

with community-acquired bacterial meningitis to investigate the roles of

common genetic variants in the complement system in outcome. By analyzing

clinical data and CSF, we identified the potential impact and functionality of a

SNP that was associated with outcome. We than validated and explored our

24407 Heckenberg.indd 112 25-02-13 11:11


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findings in an animal model of pneumococcal meningitis and investigated

whether adjuvant treatment with a monoclonal antibody targeted against this

specific complement component could improve outcome.

Methods

Dutch bacterial meningitis cohort

The nationwide prospective cohort study included patients with bacterial

meningitis that were older than 16 years of age with positive CSF cultures, who

were identified by NRLBM from March 2006 to June 2009. NRLBM provided the

names of the hospitals in which patients with bacterial meningitis had been

admitted 2–6 days previously, and the treating physician was contacted for

permission to include the patient. Controls for exposure/susceptibility were

patients’ partners or their nonrelated proxies living in the same dwelling. Data

on age, sex, and ethnicity of controls were collected. Secured online case-

record forms were used to collect data on patient history, symptoms and

signs on admission, treatment, complications, and outcome. Outcome was

graded at discharge according to the GOS, a wellvalidated instrument with

good inter-observer agreement.23 A score of 1 on this scale indicates death; a

score of 2 indicates a vegetative state; a score of 3 indicates severe disability;

a score of 4 indicates moderate disability; and a score of 5 indicates mild or no

disability. A favorable outcome was defined as a score of 5, and an unfavorable

outcome was defined as a score of 1 to 4. Blood from patients and controls

for DNA extraction was collected in sodium/EDTA. DNA was isolated with the

Gentra Puregene Isolation Kit (Qiagen), and quality control procedures were

performed to determine the yield and purity.

Genotyping

A total of 17 common SNPs in the complement system were genotyped using

TaqMan SNP Genotyping Assays (Applied Biosystems) with 96 × 96 Dynamic

Arrays (Fluidigm) by Service XS, Leiden, the Netherlands, and the Genetics Core

Facility in the Academic Medical Center. Laboratory personnel were blinded to

clinical information.

24407 Heckenberg.indd 113 25-02-13 11:11

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CSF complement analysis

CSF of patients was obtained from the diagnostic lumbar puncture.

Subsequently, CSF and wbc were stored separately at –80°C. CSF complement

component C5a and TCC levels were determined using the Microvue C5a and

sC5b-9 (TCC) Quidel ELISA Kits according to the manufacturer’s instructions.

Strength of relationships between C5a and TCC levels and clinical or biological

features was assessed by Spearman’s correlation tests.

Animal pneumococcal meningitis model

A well-characterized mouse model of pneumococcal meningitis was used in

this study.25 Prior to infection, mice were weighed and scored clinically, and

temperature was taken. For clinical scoring, different tasks were evaluated,

namely a postural reflex test and a beam walk test. Additionally, clinical

scoring comprised presence of seizures, piloerection, or reduced vigilance.57

The maximum clinical score was 12 and indicated severe disease, whereas a

score of 0 defined healthy, uninfected mice. To further evaluate locomotor and

exploratory behavior the OFT was used. In this test, mice were put in the center

of a square box, subdivided into 9 fields. Mice were observed for 2 minutes, and

the number of entered fields was counted. After clinical evaluation, bacterial

meningitis was induced by intracisternal injection of 15 μl 107 CFUs per ml S.

pneumoniae type 2 (D39 strain; provided by Sven Hammerschmidt, University of

Greifswald, Greifswald, Germany) under short-term anesthesia with isoflurane.

To evaluate the acute disease, animals were investigated 24 hours after infection.

To evaluate adjuvant treatment options, mice received antibiotic therapy (100

mg/kg ceftriaxone i.p.) together with adjuvant treatment at 24 hours after

infection and were investigated 48 hours after infection. In both settings, at

the end of each experiment, animals were weighed and scored clinically as

described above, and the temperature was taken. Mice were then anesthetized

with ketamine/xylazine, and a catheter was placed into the cisterna magna.

CSF samples were obtained for wbc count and determination of bacterial

titers. ICP was measured. Finally, animals were perfused transcardially with ice-

cold PBS, and brains were removed and either frozen immediately or fixed in

formalin. Formalin-fixed brains were subsequently embedded in paraffin for

immunohistochemistry.

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Acute mouse model of pneumococcal meningitis

The following experimental groups were investigated: (a) WT mice injected

intracisternally with 15 μl PBS (controls; C57BL/6, male, n=8 and BALB/c, male,

n=6); (b) WT mice injected intracisternally with S. pneumoniae (C57BL/6, male,

n=12; C57BL/6, female, n=20; and BALB/c, male, n=10); (c) C3ar1–/– mice

(male, genetic background C57BL/6; provided by Richard A. Wetsel, University

of Texas Health Science Center, Houston, Texas, USA) injected intracisternally

with S. pneumoniae (n=12); (d) C3a/GFAP mice (male, genetic background

C57BL/6) injected intracisternally with S. pneumoniae (n = 11); (e) C6–/–

mice (female, genetic background C57BL/6) injected intracisternally with S.

pneumoniae (n=14); (f ) Cd59a–/– mice (female, genetic background C57BL/6)

injected intracisternally with S. pneumoniae (n=11); (g) C5ar1–/– mice (male,

genetic background BALB/c, obtained from The Jackson Laboratory) injected

intracisternally with S. pneumoniae (n=9); (h) WT mice injected intracisternally

with S. pneumoniae and treated i.p. with either a neutralizing monoclonal

antibody directed against murine C5 (1 mg per mouse; clone BB5.1, n=7) 58,59 or mouse IgG antibodies (1 mg per mouse, n=12; Innovative Research);

(i) WT mice injected intracisternally with S. pneumoniae and treated i.p. with

a neutralizing monoclonal antibody directed against murine C5 (30 μg per

mouse; clone BB5.1, n=3), i.t. with a neutralizing monoclonal antibody directed

against murine C5 (30 μg per mouse; clone BB5.1, n=4), or i.t. mouse IgG

antibodies (30 μg per mouse, n=4); (j) WT mice injected intracisternally with

S. pneumoniae and treated i.p. with 250 μg anti–GR-1 (granulocyte depletion

antibody; n=8) or mouse IgG antibodies (250 μg per mouse, n=8); (k) WT mice

injected intracisternally with S. pneumoniae and treated i.p. with a neutralizing

monoclonal antibody directed against CXCL2/MIP-2 (100 μg per mouse, n=3),

the neutralizing antibody against CXCL2/MIP-2 combined with a neutralizing

antibody directed against CXCL1/KC (100 μg per mouse, n=4), rat isotype

control antibodies (IgG2B; 100 μg per mouse, n=3), or rat isotype control

antibodies (100 μg IgG2B and 100 μg IgG2A per mouse, n=4); (l) WT mice

injected intracisternally with S. pneumoniae and treated i.p. with a neutralizing

monoclonal antibody directed against murine TLR2 and TLR4, clone T2.5

(mTLR2), and clone 1A6 (hTLR4) (n=8) (provided by Novimmune) (0.75 mg each

per mouse, n=5) or mouse IgG antibodies (1.5 mg per mouse, n=5).

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Treatment mouse model of pneumococcal meningitis

WT mice were injected intracisternally with S. pneumoniae (C57BL/6, male) and

additionally treated i.p. with (a) PBS (250 μl at 24 and 32 hours after infection;

n=16); (b) dexamethasone (0.5 mg/kg at 24 and 32 hours after infection; n=10);

(c) a neutralizing monoclonal antibody directed against murine C5 (1 mg per

mouse; clone BB5.1, n=10); (d) neutralizing antibodies direct against TLR2 and

TLR4 (0.75 mg each per mouse; n=8); and (e) mouse IgG antibodies (1 mg per

mouse; n=21).

Determination of cerebellar bacterial titers

For determination of bacterial titers, the cerebellum was dissected and

homogenized in 1 ml sterile PBS. Cerebellar homogenates were diluted serially,

plated on blood agar plates, and cultured for 24 hours before CFUs were counted.

Neuroscore

For better comparison, the degree of breaching of the BBB integrity and the

number of intracerebral hemorrhages were combined in a neuroscore. For the

determination of the BB integrity, frozen mouse brain extracts were examined

for diffusion of albumin using ELISA as described previously.25 The score was

0, 1, or 2, if brain albumin was 0–35, 36–75, or 76–140 ng/μg, respectively. For

more than 140 ng/μg of albumin, the score assigned was 3. For determination

of intracerebral hemorrhage, mouse brains were cut in a frontal plane into 10-

mm thick sections. Beginning from the anterior parts of the lateral ventricles,

9 serial sections were photographed with a digital camera at 0.3-mm intervals

throughout the ventricle system. Hemorrhagic spots were counted, and the

bleeding area was measured. A score of 0 indicates no cerebral bleedings, a

score of 1 indicates up to 20 cerebral bleeding spots, a score of 2 indicates

between 21 and 60 cerebral bleeding spots, and a score of 3 indicates more

than 60 cerebral bleeding spots. The maximum neuroscore was 6 and indicated

severe neuronal damage, whereas a score of 0 indicated no neuronal damage.

Analysis of protein expression

Expression of C5a, TCC, IL-6, CXCL1/KC, and CXCL2/MIP-2 was determined

in mouse brain homogenates by ELISA according to the manufacturer’s

instructions (C5a and TCC, USCN Life Science, Biozol; IL-6, CXCL2/MIP-2, and

CXCL1/KC, R&D Systems). Expression profiles of C5a and TCC were additionally

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evaluated by immunohistochemistry performed on paraffin-embedded slides

of mouse brain tissue as previously described.60 Briefly, after deparaffinization

and steam bath antigen retrieval in citrate buffer, endogenous peroxidase was

quenched with 7.5% hydrogen peroxide. Nonspecific binding was minimized

by incubation in 10% normal goat serum. Slides were then incubated

overnight at 4 °C with a rat anti-mouse C5a or TCC antibody or the appropriate

isotype control immunoglobulin. Specific labeling was detected with a biotin-

conjugated rabbit anti-rat antibody and application of horseradish peroxidase–

bound avidin/biotin from Vectastain ABC Kits, followed by development

with 3,3’-diaminobenzidine (DAB) solution (both from Vector Laboratories).

Counterstaining was performed using Mayer’s hematoxylin. Slides were

digitized using a Zeiss Axiovert microscope (Carl Zeiss) connected to a cooled

Moticam 5000 video camera (Moticam).

Statistical analysis – genetic analysis

For evaluating the role of SNPs on outcome, assuming an overall event rate

of 25% (n=100 cases) to patients with favorable outcome (n=300), a sample

size of 400 provides sufficient power (80%) when a risk genotype has a relative

risk of 3.0 or more, using a P value of 0.0029 (Bonferroni corrected). The Mann-

Whitney U test was used to identify differences in baseline characteristics

among groups with respect to continuous variables, and dichotomous

variables were compared with use of the χ2 test. These statistical tests were

2-tailed, and a P value of less than 0.05 was regarded as significant. Differences

in genotype frequencies were analyzed with the χ2 or Fishers’ exact tests by

use of the programs R-statistics and PASW18. For the SNP analysis, we used a

Bonferroni correction for multiple testing (17 SNPs; p<0.0029). We calculated

whether the genotype frequencies in the control groups concurred with the

HWE by use of a χ2 and exact test with 1 degree of freedom with a p-value

of less than 0.05 to indicate significance. SNPs deviating from the HWE were

excluded. The genotype frequencies of patients with a favorable outcome

was compared with those with an unfavorable outcome as defined by the

GOS. Subgroup analyses were defined by ethnicity (mixed European descent),

causative organism (S. pneumoniae), and a combination of these factors. We

used a multivariate logistic regression analysis to calculate ORs and 95% CIs to

assess the strength of the association among potential risk factors (including

identified polymorphisms) and outcome.

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Statistics – animal experiments

The principal statistical test was a 2-tailed unpaired Student’s t test (combined

with an α-adjustment in case of multiple comparisons) or a logrank test

(Mantel) for survival. Differences were considered significant at p<0.05. Data

are displayed as mean±SD.

Study approval

The protocol used in this study was approved by the Academic Medical Center

and all local participating hospitals (see Supplementary Methods). Written

informed consent was obtained from all participating patients, or their legally

authorized representatives, and controls. All animal experiments were approved

by the animal ethic committee of the government of Upper Bavaria, Germany.

Results

Nationwide prospective cohort study of adults with community-acquired bacterial

meningitis

In a prospective nationwide cohort study, we included 62 out of 762 (84%)

identified episodes of communityacquired CSF culture-proven bacterial

meningitis in 636 patients. The distribution of causative bacteria was S.

pneumoniae in 468 (73%), N. meningitidis in 80 (13%), and other bacteria in 94

(15%) episodes. DNA samples were obtained from 439 patients (68%) and 302

controls. Controls were patients’ partners or nonrelated proxies living in the

same dwelling, as household members they had similar exposure to bacteria

through nasopharyngeal colonization, and were matched for age, ethnicity,

and sex (ref. 22 and Supplementary Table 7.1; supplementary material available

online with this article; di:10.1172/JCI57522DS1). Predisposing conditions,

most commonly otitis media or sinusitis (36%) and immunocompromised

state (22%), were present in 58% of episodes (Table 1). In 13% of episodes,

patients were comatose on admission, and 32% of the episodes had focal

neurologic deficits. The case fatality rate was 8%, and 24% of the episodes had

an unfavorable outcome, defined as a score of 1 through 4 on the Glasgow

Outcome Scale (GOS).23 Patients for whom DNA was obtained were on average

ounger and presented with less severe disease than patients for whom DNA

was not obtained (Supplementary Table 7.2).

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Table 1. Clinical characteristics of 439 patients with community-acquired bacterial meningitisa

Characteristic Value

Age – yr 56 ±18

Male sex – no (%) 208 (47%)

Duration of symptoms <24 hr 200/436 (46%)

Pretreatment with antibiotics 51/433 (12%)

Predisposing conditions 253/436 (58%)

Otitis or sinusitis 156/436 (36%)

Pneumonia 57/436 (13%)


Symptoms and signs on presentationb

Headache 340/394 (86%)

Neck stiffness 325/421 (77%)

Systolic blood pressure – mmHg 145 ±29

Heart rate – beats/min 99 ±21

Body temperature – º C 38.7 ±1.3

Score on Glasgow Coma Scalec 11 ±3

<8 indicating coma 58/434 (13%)

Focal neurologic deficits 140/436 (32%)

Indexes of CSF inflammationd

Opening pressure (mm H2O) 387 ±126

White blood cell count (/mm3) 6708 ±11964

<1000/mm3 116/409 (28%)

Protein – g/L 4.3 ±3.1

CSF: blood glucose ratio 0.15 ±0.16

Positive blood cultures 273/365 (75%)

Complications

Cardiorespiratory failure 118/420 (28%)

Focal neurologic deficits 86/425 (20%)

Cerebral infarction 50/436 (11%)


1 – death 35/435 (8%)

2 – vegetative state 1/435 (0.2%)

3 – severe disability 15/435 (3%)

4 – moderate disability 55/435 (13%

5 – good recovery 329/435 (76%)a Data are number/number evaluated (percentage), continuous data are mean ±SD. b Systolic blood pressure was evaluated in 426 patients, heart rate in 421, temperature in 432. c CSF opening pressure was evaluated in 151 patients, CSF WBC count in 409 patients, CSF protein in 412, CSF blood: glucose ratio in 408.

24407 Heckenberg.indd 119 25-02-13 11:11

Chapter 7

120

Tab

le 2

. Gen

otyp

ing

anal

ysis

of

17 c

omm

on c

omp

lem

ent

com

pon

ent

pol

ymor

phi

sms

in 3

29 b

acte

rial m

enin

gitis

pat

ient

s w

ith f

avor

able

out

com

e an

d 10

5 w

ith

unfa

vora

ble

out

com

e

Favo

rab

le o

utco

me

Unf

avor

able

out

com

eRi

sk a

llele

Gen

eSN

P ID

AB

AA

AB

BBA

BA

AA

BBB

or

geno

typ

eO

R (9

5% C

I)P

- val

ue

C3rs

1047

286

543

111

218

107

216

937

6931

3BB

4.88

(0.8

0-29

.6)

0.09

2

C3rs

2230

199

539

115

218

103

617

133

7325

4BB

2.18

(0.6

0-7.

90)

0.22

3

C5rs

1761

129

236

665

162

102

7713

118

4145

BB1.

70 (1

.08-

2.67

)0.

021

C6rs

1801

033

422

232

140

142

4513

767

4547

10A

1.47

(0.7

1-3.

03)

0.29

7

C7rs

1063

499

257

401

5714

312

990

114

2344

35A

A1.

39 (0

.81-

2.40

)0.

236

C7rs

1315

7656

174

472

1215

016

149

151

539

56BB

1.28

(0.8

2-2.

01)

0.28

2

C7rs

6071

4178

9156

58

7524

534

176

524

76A

A2.

00 (0

.64-

6.25

)0.

225

C8Ba

rs12

0675

0735

621

819

301

2118

57

789

AA

2.92

(1.0

3-8.

26)

0.03

5

C8B

rs12

0854

3562

428

298

280

185

1786

132

BB1.

02 (0

.99-

1.05

)0.

056

C9rs

7002

3338

625

011

415

846

110

7832

4616

BB1.

21 (0

.65-

2.26

)0.

543

C9rs

3488

2957

607

4928

145

219

210

9110

0A

A1.

52 (0

.74-

3.13

)0.

252

CFH

rs50

5102

456

198

161

134

3214

559

5241

9A

1.12

(0.5

2-2.

43)

0.77

3

CFH

rs10

6548

910

754

916

7523

738

168

626

71A

1.17

(0.7

2-1.

90)

0.51

5

CFH

rs14

1099

635

030

892

166

7111

195

3149

23A

A1.

11 (0

.68-

1.80

)0.

675

CFH

rs37

5339

610

055

88

8423

732

174

228

73B

1.26

(0.2

6-6.

02)

0.56

0

CFH

rs66

7760

451

314

319

711

912

163

4364

354

AA

1.09

(0.6

9-1.

72)

0.70

7

CFH

rs37

5339

415

550

320

115

194

6414

213

3852

AA

2.23

(1.0

7-4.

65)

0.02

9

CFH

rs10

6117

043

422

414

813

843

137

7350

3718

BB1.

38 (0

.76-

2.51

)0.

296

For f

avor

able

and

unf

avor

able

out

com

e, th

e nu

mb

er o

f pat

ient

s in

eac

h al

lele

or g

enot

ype

is li

sted

. a Con

trol

pop

ulat

ion

did

not c

omp

ly w

ith H

WE.

24407 Heckenberg.indd 120 25-02-13 11:11


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Genetic association study on common variants in the complement system

We selected all SNPs with a minor allele frequency of more than 5% in genes

coding for complement components (C1QA, C1QB, C1QC, C2, C3, C5, C6, C7,

C8B, C9, CFD, CFH, CFI, and CFP) for which a commercial genotyping assay was

available. A total of 17 SNPs were genotyped using TaqMan SNP Genotyping

Assays (Applied Biosystems). The genotyping success rate was more than

95% for all assays. In 16 out of 17 assays, genotype frequency of controls of

mixed European descent concurred with the Hardy-Weinberg equilibrium

(HWE; Supplementary Table 7.3). We compared the genotype frequency of

patients with a favorable outcome, defined as a GOS score of 5, indicating mild

or no disability, with that of patients with an unfavorable outcome. Using a

Bonferroni correction for multiple testing, we identified rs17611 in complement

component 5 (C5; GG genotype) to be associated with unfavorable outcome in

patients of mixed European descent with pneumococcal meningitis (OR, 2.25;

95% CI, 1.33–3.81; p=0.002).

In a multivariate regression analysis, including previously identified important

risk factors for unfavorable outcome (age, CSF wbc count <1,000/mm3, score

on the Glasgow Coma Scale, blood thrombocyte count, immunocompromise,

otitis media, and/or sinusitis), the predictive effect of rs17611 remained robust

(OR, 1.92; 95% CI, 1.09–3.26; p=0.032; Supplementary Table 7.4).3 Other SNPs

frequencies were similar in patients with unfavorable and favorable outcome

(Tables 2 and 3).

Complement in CSF of adults with bacterial meningitis

C5-convertase cleaves C5 into the anaphylatoxin C5a and fragment C5b. When

C5b associates with C6 and C7, the complex becomes inserted into bacterial

membranes and interacts with C8, permitting the binding of several copies

of C9 to form the MAC.12 To explore the role of C5 in patients with bacterial

meningitis, we measured CSF levels of C5a and terminal complement complex

(TCC; sC5b-9) in the CSF of 204 out of 642 episodes, using the Quidel Microvue

C5a and sC5b-9 ELISA Kits. Baseline characteristics and outcome were similar

for patients with CSF available as compared with those of patients without CSF

available. C5a and TCC levels were correlated with Glasgow Coma Scale scores

on admission, death, and unfavorable outcome (Figure 1). Higher levels of C5a

and TCC predicted increased parameters of CSF inflammation. There was no

significant association between CSF C5a or TCC levels and rs17611 genotypes

24407 Heckenberg.indd 121 25-02-13 11:11

cHapter 7

122

(C5a, 8.7 ng/ml [interquartile range, IQR, 2.0–43] in rs17611A vs. 16 ng/ml [IQR,

4.0–64] in rs17611GG, p=0.29; TCC, 2.0 μg/ml [IQR, 0.3–4.4] in rs17611A vs. 2.3

μg/ml [IQR, 0.5–5.5] in rs17611GG, p=0.50).

Figure 1. Association of C5a and TCC concentrations in CSF with disease severity and outcome

Glasgow Coma Scale score Glasgow Coma Scale score

CSF protein (g/L) CSF protein (g/L)0 2 4 6 8 10 12 14

3

200

150

100

50

0

200

150

100

50

0 0

0

10

10

20

20

15

15

5

5

5 7 9 11 13 15

)L/gn( FSC a5

C) L/ gn( FS

C a5C

( FSC

CCT

µ)L/g

( FSC

CCT

µ) L/ g

Co-eff -.201, P = 0.004Co-eff -.196, P = 0.018

Co-eff 0.466, P < 0.001 Co-eff 0.631, P < 0.001

A

C D

B

CSF WBC>1000/mm3

Unfavorable outcome

Dead CSF WBC>1000/mm3

Unfavorable outcome

Dead

)L/gn( FSC a5

C

( FSC

CCT

µ)L/g

0

2

4

6

0

20

60

40 P = 0.09P = 0.001

P = 0.011

P = 0.001

P = 0.001

P = 0.03

0 2 4 6 8 10 12 14

FFFE

Co-eff -.201, P = 0.004

Glasgow Coma Scale score1514131211109876543

TCC

CSF

(ng/

ml)

20000

15000

10000

5000

0

Co-eff 0.466, P < 0.001

CSF Protein (g/L)14,012,010,08,06,04,02,00,0

C5a

CSF

(ng/

ml)

200

150

100

50

0

Co-eff 0.631, P < 0.001

CSF Protein (g/L)14,012,010,08,06,04,02,00,0

TCC

CSF

(ng/

ml)

20000

15000

10000

5000

0

Co-eff -.196, P = 0.018

Glasgow Coma Scale score1514131211109876543

C5a

CSF

(ng/

ml)

200

150

100

50

0

Page 1

3 5 7 9 11 13 153 5 7 9 11 13 15

8 10 12 14

Co-eff -.196, P = 0.018 Co-eff -.201, P = 0.004

Co-eff 0.466, P < 0.001 Co-eff 0.631, P < 0.00120

15

10

5

0

Association of C5a and TCC concentrations in CSF with disease severity and outcome. (A–D) Pearson correlation analysis of C5a (A and C) and TCC (B and D) CSF levels with Glasgow Coma Scale score and CSF protein concentration. Each dot represents an individual patient; diagonal lines represent the mean. Co-eff , coeffi cient. (E and F) Median (E) C5a and (F) TCC CSF levels in patients with CSF wbc counts of more than 1,000 (white bars) versus those with less than 1,000 (black bars), unfavorable (white bars) versus favorable outcome (black bars), and deceased (white bars) versus surviving patients (black bars). P values for diff erences between groups were determined with the Mann-Whitney U test.

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Patients with pneumococcal meningitis with the rs17611 GG genotype had

lower CSF wbc counts (2,185 per mm3 [IQR, 375–7,738] vs. 3,956 per mm3

[IQR, 998–9,365]; p=0.036) but similar CSF protein and CSF glucose levels,

as compared with those of patients with AA or AG alleles. Lower CSF wbc

counts have been reported to predict unfavorable outcome in patients with

community-acquired bacterial meningitis. To obtain insight into the functional

role of terminal complement components in bacterial meningitis, we next

performed experiments using a mouse model of pneumococcal meningitis. 25

Expression profile of C5a and the TCC C5b-9 in the mouse model of pneumococcal

meningitis

To confirm that C5a and the TCC are expressed in the meningitis mouse

model, we examined mouse brain homogenates from WT mice infected with

S. pneumoniae. At 24 and 48 hours after infection, C5a and TCC levels were

increased (Figure 2A and 2B). Immunohistochemical staining was positive for

C5a and TCC in and around inflammatory infiltrates in brains of infected mice

(Figure 2C and 2D). TCC expression was also detected in cortical vessels (Figure

2D).

Functional analysis of C5a, MAC, and C3 in the mouse model of pneumococcal

meningitis

Next, we examined the functional role of the anaphylatoxins, C5a and C3a,

and MAC in our mouse model using different mutants. Components of the

complement system are known to modulate inflammatory responses.12, 26 First,

we compared mice with a deficiency of the C5a receptor (C5ar1–/– mice) to WT

mice. CSF wbc count in infected C5ar1–/– mice was decreased to 25% of that in

WT mice (Figure 3). The reduced inflammatory response in C5ar1–/– mice was

associated with better clinical status (clinical score shown in Figure 3), with less

severe hypothermia, reduced weight loss, and conserved exploratory behavior

in the open-field test (OFT) (C5ar1–/– vs. WT, body temperature, 37.3°C ± 0.42°C

vs. 36.6°C ± 0.54°C, p=0.004; weight loss, 11.1% ± 1.58% vs. 13.3% ± 2.38%,

p=0.031; OFT, 23 ± 22 fields vs. 3 ± 4 fields, p=0.019). A strong granulocytic

inflammatory response contributes substantially to neuropathology in

pneumococcal meningitis; this was supported by our finding that granulocyte

depletion was protective against meningitis-related brain damage

(Supplementary Figure 7.1).27 Therefore, we evaluated major meningitis-

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associated intracranial complications in our model: raised intracranial

pressure (ICP), decreased blood-brain barrier (BBB) integrity, and intracerebral

hemorrhages. BBB breakdown (brain albumin content) was combined with the

number of intracerebral hemorrhages to obtain the neuroscore, as described

previously.25

Figure 2. Expression profile of the anaphylatoxin C5a and the TCC in mice with pneumococcal meningitis

(A) Levels of C5a (ng/mg) and (B) TCC (ng/mg) were determined by ELISA in brain homogenates of WT mice intracisternally injected with PBS (controls, n=4) or infected with S. pneumoniae at different time points after infection (6, 24, and 48 hours; n=5 each) (above). Levels of C5a and TCC were both found to be increased at 24 hours (*p=0.006 and † p=0.002) and 48 hours (**p=0.014 and †† p=0.016) after infection (unpaired Student’s test and Bonferroni correction for multiple measurements; data are shown as mean±SD). (C and D) Formalin-fixed and paraffin-embedded brains of WT mice infected for 24 hours were used for immunohistochemistry. C5a and TCC immunoreactivity was visualized with streptavidin horseradish peroxidase and DAB, which yields a brown reaction product. In infected mice, positive immunostaining was seen in the inflammatory infiltrates for both C5a (original magnification, ×400 [C]) and TCC (original magnification, ×1,000 [D]).

C5ar1–/– mice had reduced ICP and lower neuroscores when compared with

those of WT mice (Figure 3). There was no difference in cerebellar bacterial

titers (6.58 ± 0.59 log10 CFUs/cerebellum in WT mice vs. 6.15 ± 0.73 log10 CFU/

cerebellum in C5ar1–/– mice) or mortality rate (0 out of 10 vs. 0 out of 9 for WT

vs. C5ar1–/– mice) between C5ar1–/– and WT mice. The altered recruitment of

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CSF inflammatory cells in C5ar1–/– mice prompted us to analyze the levels of

cytokines and inflammatory mediators in mouse brain homogenates. Amounts

of IL-6 (data not shown), CXCL1/KC, and CXCL2/MIP-2 were all reduced in

infected C5ar1–/– mice compared with those in infected WT mice (Figure 3).

Figure 3. Role of C5a in the mouse model of pneumococcal meningitis

To evaluate the role of C5a, C5ar1–/– mice (n=9) were examined. Animals were infected with S. pneumoniae and evaluated at 24 hours after infection for CSF leukocyte count (CSF wbc count), clinical score, ICP, BBB breaching and intracerebral hemorrhage combined in the neuroscore, and expression of proinflammatory mediators and cytokines, namely CXCL1/KC and CXCL2/MIP-2. Infected C5ar1–/– mice were compared with infected WT mice (BALB/c, male; n=10). BALB/c mice intracisternally injected with PBS served as controls (BALB/c controls; n=6). Compared with infected WT mice, C5ar1–/– mice displayed reduced CSF leukocytosis (*p=0.001) accompanied by a better clinical status (†p=0.001) and reduced secondary CNS complications (reduced ICP [‡p=0.001] and neuroscore [**p=0.025]). Levels of IL-6, CXCL1, and CXCL2 were reduced in C5ar1–/– mice (p= 0.038, ††P=0.019, and ‡‡ p=0.047, respectively; unpaired Student’s test; data are shown as mean±SD).

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In order to evaluate whether the decreased CSF wbc count observed in the C5ar1–

/– mice was mediated through chemokine regulation by C5a, infected animals

were treated with anti-CXCL2/MIP-2 antibodies, either alone or in combination

with anti-CXCL1/ KC antibodies. Treatment with anti-CXCL2/MIP-2 antibodies

alone reduced CSF wbc count by 40% (not significant), whereas, when combined

with anti-CXCL1, treatment caused a reduction of CSF wbc count by 63%

(p=0.003). The animals treated with both CXCL1 and CXCL2 antibodies were in

a better clinical state compared with that of the untreated mice (Supplementary

Table 7.5). To analyze the role of the MAC, we investigated mice with a mutation

in the complement component 6 gene (C6–/– mice), which are unable to form

the MAC, and mice gene deleted for CD59 (Cd59a–/– mice), the in vivo inhibitor

of the MAC (12). C6–/– mice with pneumococcal meningitis tended to have lower

CSF wbc counts as compared with those of WT mice (Supplementary Figure

7.2), whereas Cd59a–/– mice had increased CSF wbc counts compared with

those of WT animals (Supplementary Figure 7.3). No differences were detected

between C6–/–, Cd59a–/–, and WT mice in clinical scores, ICP, or neuroscores

(Supplementary Figure 7.3). However, the mortality rate among C6–/– mice was

higher (7 out of 14 [50%]) compared with that of Cd59a–/– mice (2 out of 11

[18%]) and WT mice (2 out of 20 [10%]). This difference was attributable to a more

severe damage of the BBB in C6–/– mice compared with that of WT mice (brain

albumin content, 487.0 ± 287.7 ng/μg vs. 242.7 ± 178.0 ng/μg, p=0.014). Levels of

IL-6 and CXCL2/MIP-2 were similar among the 3 mouse strains.

We next investigated the role of C3a in pneumococcal meningitis. The

anaphylatoxin C3a has been shown to be involved in immune regulation of

inflammatory CNS diseases, and we previously described increased expression

of the C3a receptor in mice with pneumococcal meningitis.21, 28 Mice deficient

in the C3a receptor (C3ar1–/– mice) and mice expressing C3a exclusively in the

CNS using the GFAP promoter (C3a/GFAP mice) were compared with infected

WT mice. C3a/GFAP mice had increased CSF wbc counts as compared with those

of WT and C3ar1–/– mice (Supplementary Figure 7.3), but other parameters

were similar (clinical scores, ICP, neuroscores, proinflammatory mediators, and

cytokines; Supplementary Figure 7.3).

Adjuvant treatment with C5 antibody.

The experiments performed with C5ar1–/– mice suggested a major role for

C5a in the regulation of the immune response in pneumococcal meningitis;

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we therefore evaluated treatment with a neutralizing monoclonal antibody

directed against murine C5 (C5-Ab, BB5.1) in the model. Animals were given i.p.

C5-Ab or i.p. IgG (1 mg per mouse, each) prior to infection. Levels of cerebral

sC5b-9 were significantly reduced in animals treated with C5-Ab (Figure 4).

Consistent with that in C5ar1–/– mice, WT mice treated with C5-Ab prior to

infection displayed a reduced CSF wbc count accompanied by better clinical

scores when compared with those of animals treated with mouse IgG (Figure

4). To define the site of action of C5 neutralization, we applied a low dose of C5-

Ab (30 μg per mouse) to infected mice either by the i.p. (systemic) or intrathecal

(i.t.) (local) route. Mice treated i.t. with C5-Ab had lower CSF leukocyte counts,

less meningitis-associated intracranial complication, and better clinical status,

as compared with mice treated with control IgG (Supplementary Table 7.5). No

difference between infected mice treated with i.p. C5-Ab or control IgG was

observed. We next compared adjunctive C5-Ab treatment with adjunctive

treatment with dexamethasone, the standard adjunct in humans with

pneumococcal meningitis, or adjunctive treatment with neutralizing TLR2 and

TLR4 antibodies.1, 8 Treatment with TLR2 and TLR4 antibodies was based on our

recent observation that TLR2 and TLR4 are essential in mounting the CNS innate

immune response in pneumococcal meningitis. 29 All adjunctive therapies were

administered i.p. 24 hours after infection concomitant with antibiotic treatment

consisting of ceftriaxone. In these experiments, i.p. treatment with PBS or IgG

served as control. Treatment with the C5-Ab prevented lethal outcome in all

treated animals, as shown by a significant decrease in the mortality rate as

compared with treatment with IgG (deaths, 7 out of 21 mice [33%]; Figure

5A). Adjunctive treatment with dexamethasone reduced the mortality rate

as compared with that with PBS (deaths, 2 out of 10 mice [20%] vs. 5 out of

16 mice [31%]) but was less effective when compared with treatment with

C5-Ab (Figure 5C). Adjunctive treatment with anti-TLR2 and TLR4 antibodies

caused a significant attenuation of meningeal inflammation and brain tissue

damage, in line with our previous study (Supplementary Table 7.5); however,

these antibodies had no effect on mortality (deaths, 2 out of 8 mice [25%];

Figure 5B).29 Adjunctive treatment with C5-Ab, but not with dexamethasone or

anti-TLR2 and 4 antibodies, resulted in a reduction of meningitis-induced brain

damage (neuroscores, 2.3 ± 1.6 vs. 4.2 ± 1.6 in IgG-treated mice [p=0.012], vs. 4.3

± 1.5 in anti-TLR2– and TLR4–treated mice, 3.5 ± 2.0 in dexamethasonetreated

mice, and 3.7 ± 1.7 in PBS-treated mice).

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Figure 4. Pretreatment model of C5-Ab

To confirm the activity of C5-Ab, we first treated animals with i.p. C5-Ab (n = 7) or i.p. IgG (n = 12) prior to infection. After 24 hours, expression of the TCC C5b-9 was determined in mouse brain homogenates (left). Additionally, animals were evaluated for CSF leukocyte count (CSF wbc count; middle) and clinical score (right). Animals pretreated with the antibody to C5 prior to infection displayed reduced levels of C5b-9 (*P = 0.012) and reduced CSF leukocytosis (†P = 0.001) accompanied by a better clinical status (‡P = 0.002). Unpaired Student’s test; data are shown as mean ± SD.

Figure 5. Effect of additional treatment with a monoclonal antibody to murine C5 or with dexamethasone on survival in mice with pneumococcal meningitis

Additional treatment with a monoclonal antibody to (A) murine C5 (anti–C5-Ab; n=10), (B) antibody to TLR2 and TLR4 (anti-TLR2/4; n=8), or (C) dexamethasone (n=10) was administered 24 hours after infection together with antibiotic treatment with ceftriaxone. Administration of an IgG1 isotype control (IgG1; n=21) or PBS (n=16) served as control. Kaplan Meier curves of survival are shown. Additional treatment with C5-Ab prevented lethal outcome in all animals (p=0.047). Adjuvant therapy with dexamethasone or with anti-TLR2 and TLR4 antibodies had no significant effect on meningitis-associated death (death rate, 20% or 25% compared with 31% in PBS-treated animals). (D) Representative brain sections obtained from mice from the different experimental groups 48 hours after infection. Only treatment with C5-Ab led to a visible reduction in cerebral hemorrhages observed in infected mice treated with either control IgG or the vehicle PBS.

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Discussion

We demonstrated that a common variant in C5 was associated with unfavorable

outcome in adults with community-acquired pneumococcal meningitis.

The anaphylatoxin C5a was identified as the crucial complement product in

pneumococcal meningitis. Neutralization experiments showed that adjunctive

treatment with C5-Ab improved outcome in mice with pneumococcal

meningitis. The observed effect of C5-Ab was superior to that of adjuvant

dexamethasone, the antiinflammatory drug that is currently recommended in

clinical guidelines.2, 30 Since anti-C5 antibodies are currently licensed for clinical

use (eculizumab) or used in clinical trials (pexelizumab)31, 32, our results present

a promising treatment option for future patients with communityacquired

bacterial meningitis.

Patients with the rs17611 GG genotype were at higher risk for unfavorable

outcome as compared with carriers of the A allele (OR, 2.26; 95% CI, 1.30–3.94).

Our genetic association study was nationwide, and, therefore, we were able

to study a representative sample of adults with acute bacterial meningitis.

The prospective approach allowed us to collect comprehensive clinical data,

resulting in a well-defined group of patients with microbiologically confirmed

community-acquired bacterial meningitis. Our large sample gave us the

statistical power to perform a Bonferroni correction for multiple testing, and,

subsequently, we were able to validate our findings in a mouse model of

pneumococcal meningitis. Patients with the rs17611 risk genotype GG had

lower CSF wbc counts on admission. Clinical studies have shown that lower CSF

wbc counts on admission in patients with bacterial meningitis are associated

with sepsis and systemic compromise and adverse outcomes later in disease

course.3,33 Sepsis was not more common in patients with the GG genotype

in this study, although power may be insufficient to detect such a difference.

Animal studies in a pneumococcal meningitis model showed that lower CSF

wbc counts early in disease course were associated with high bacterial load,

which correlates with intracranial complications and poor outcome.34 These

experiments also showed that later in disease course, higher CSF wbc counts

correlated with high bacterial loads and were associated with poor outcome.34

Other experimental work in pneumococcal meningitis showed a critical role for

the cumulative exposure to bacteria during the infection period.35 We speculate

that the lower CSF wbc counts in patients with the risk genotype may be due to

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a reduced chemoattractant function of C5a. Functional studies have previously

shown that SNPs in complement factors can influence complement activation

and binding affinity independent of concentration.36,37 A study on rs17611

function showed the GG genotype was associated with reduced serum C5

concentration among 100 healthy volunteers.38 A follow-up study, however,

showed that these subjects had serum C5 activity similar to that of those with

rs17611 AA/AG, despite lower C5 serum concentration.39 This observation is

consistent with our results, which showed similar C5a and TCC concentration

in both genotypes. The anaphylatoxin C5a is a powerful chemoattractant,

guiding neutrophils but also directly stimulating the production of cytokines,

chemokines, and adhesion molecules.12, 40

Major neurologic complications in patients with pneumococcal meningitis

include cerebrovascular complications and brain edema, which are caused, at

least partly, by massive neutrophilic inflammatory reaction. In patients with

bacterial meningitis, CSF C5a concentrations were markedly elevated, and C5a

levels were associated with high CSF wbc counts and unfavorable outcome.

In our mouse model, deficiency of the receptor for C5a led to an improved

clinical status and clinical course. C5a receptor deficiency and C5 neutralization

resulted in a marked reduction of CSF wbc counts in the pneumococcal mouse

model, with lower concentrations of IL-6, CXCL1, and CXCL2 in C5ar1–/– mice.

Pretreatment with CXCL1 and CXCL2 antibodies caused a reduction of CSF wbc

count, but to a lesser extent than that found in C5ar1–/– mice, indicating that

C5a regulates chemokine expression but also has a direct chemotactic effect.

In our experiments, i.t. anti-C5 treatment also led to a significant reduction in

CSF pleocytosis.

Previous work showed that treatment with antibodies to native human C5

inhibited leukocyte influx in rabbits with pneumococcal meningitis40, and

intracisternal administration of C5a caused rapid influx of wbc into the CSF

of rabbits.41 C5a-mediated neutrophilic inflammation may cause direct

tissue injury by release of cytotoxic products from neutrophils and/or by

precipitating cerebral vasculitis and a subsequent reduction in blood supply to

the rain.27 This concept is supported by evidence resented here and in previous

studies demonstrating that neutrophil depletion approaches are beneficial

in pneumococcal meningitis, particularly when used as adjunctive treatment

with antibiotic therapy.27, 42 These data seem to contradict our observation in

humans that the rs17611 risk genotype GG had lower CSF wbc count; however,

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CSF wbc counts were determined in samples withdrawn on admission, early in

the course of the disease. Bacterial titers were not determined in our patients;

nevertheless, it is noteworthy that the inoculum size does not correlate with

subsequent bacterial titers but does determine the disease kinetics. As a

consequence, the precise classification of disease stage is not possible in

patients with pneumococcal meningitis.

The role of C5a is not limited to its chemoattractant and proinflammatory

function. First, C5a can induce the expression of tissue factor and plasminogen

activator inhibitor-1, leading to amplification of coagulation and inhibition

of fibrinolysis.43, 44The relation between C5a and coagulation pathways

is reciprocal: thrombin directly cleaves C5 and generates active C5a, and

thrombin-activatable carboxypeptidase B inhibits C5a.43, 45 The procoagulant

activity of C5a may represent an additional and/or additive factor in the vascular

occlusion process in bacterial meningitis.24, 46, 47 Second, C5a increases vascular

permeability, thereby contributing to meningitis-induced brain edema. In

our experiments, C5a receptor deficiency and C5 neutralization resulted in a

reduction of brain albumin concentrations, indicative of a protective effect

against meningitis-induced BBB breakdown. In line with this finding is the recent

observation that C5a receptor inhibition maintained the integrity of the BBB in

experimental lupus.48 Moreover, silencing of the C5ar1 gene with siRNA was

found to prevent the bacterial lipopolysaccharideinduced increased vascular

permeability in multiple organs.49 Finally, very high concentrations of C5a were

shown to induce rapid apoptosis in neuronal cells via neuronal C5a receptor–

associated signal transduction pathways50, whereas in lower concentrations,

C5a inhibited apoptosis, induced neuroproliferation, and decreased glutamate

excitotoxicity.51 These findings imply that C5a may function as a direct

modulator of brain tissue injury in pneumococcal meningitis.

Adjunctive treatment with C5-Ab resulted in a reduction in meningitis-

induced brain damage and prevented death, despite having no effect on

either bacterial outgrowth in the CSF and blood or antibiotic-induced bacterial

killing in experimental pneumococcal meningitis. Complement-mediated

opsonophagocytosis and not MAC-mediated bacterial lysis is the major host

defense mechanism against invasive pneumococcal infections. In contrast,

MAC is known to play a major role in meningococcal killing. Anti-C5 antibodies

that block C5a and MAC formation were found to interfere with bacterial lysis

using a human whole blood model of meningococcal sepsis.52 However, this

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study also showed that C5a-specific antibodies (monoclonal antibody 137-

126) can bind the C5a moiety and inhibit the harmful effects of C5a while

preserving MAC-mediated bacterial killing.52 The observed adjuvant effect of

C5-Ab was superior to that of neutralizing antibodies against TLR2 and TLR4,

2 pattern recognition receptors (PRRs) that have been shown to be essential

for mounting the innate immune response to pneumococcal infection of

the CNS in experiments using Tlr2–/–, Tlr4–/–, and Tlr2/4–/– mice.27, 29 Indeed,

in our neutralization experiments, antibodies to both TLR2 and TLR4, when

administered prior to infection, produced a similar phenotype to that seen in

the receptor-deficient animals with reduced CSF pleocytosis and improved

brain pathology. Our data show that TLR signaling is vital for the initial innate

immune response but dispensable for the maintenance of inflammation in

meningitis during the later disease course. The presence of S. pneumoniae

in the subarachnoid space is initially recognized by TLR2 and TLR4 as well as

other PRRs. Activation of TLR2 and TLR4 by pneumococci leads to MyD88-

dependent induction and activation of the complement system in the

brain.29 Among the complement components produced, C5 and its activation

product C5a have now been singled out to be crucial for the propagation of

the inflammatory reaction. The C5a-driven inflammatory reaction, in turn,

contributes substantially to meningitis-induced vascular and tissue injury, thus

representing a major determinant for the outcome of the disease.

Our study has some limitations. A selection bias was introduced since DNA was

not available for a considerable proportion of patients (32%), particularly those

with more severe disease. Inclusion of patients with less severe disease will

decrease study power, resulting in type II errors. However, this will not negate

the association of rs17611 with outcome. The nationwide design allowed us to

detect this selection bias.11, 53 Furthermore, there may be functional differences

between the complement systems of humans and mice. Animal models in

rheumatoid arthritis showed a beneficial effect of C5a receptor blockage,

but a clinical trial showed no benefit. 54, 55 However, bacterial opsonization by

mouse complement is known to be similar to the human situation.56 Therefore,

we believe that our model is valid and provides valuable information on

complement function in pneumococcal meningitis. Overall, we have used

a clinical-based approach to generate a hypothesis that was subsequently

confirmed in animal studies.

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Acknowledgments

We thank B. Angele for her technical assistance and Sven Hammerschmidt,

University of Greifswald, Germany, for providing pneumococcal strains. We

thank M.T. van Meegen and E. Jansen for their work on genotyping.

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Supplementary Table 7.1. Baseline characteristics of patients and controls

Characteristic Patients with DNA (n=439 )

Controls (n=302)

Age (median- IQRa) 59 (41-68) 58 (45-66)

Male sex 210 (48%) 148 (49%)

Ethnicity

White 415 (94%) 287 (95%)

African 17 (4%) 13 (4%)

Asian 7 (2%) 2 (1%)aIQR – interquartile range

Supplementary Table 7.2. Baseline characteristics of included patients with and without DNAa

Characteristic Patients with DNA (n=439 )

Patients without DNA (n=197)

p-value

Age 59 (41-68) 62 (49-72) 0.001

Male sex 210 (48%) 94 (48%) 0.430


37/165 (20%) 0.915

Distant focus of infection 200/436 (46%) 73/165 (44%) 0.721

Clinical signs and symptoms

Headache 340/394 (86%) 111/134 (83%) 0.327

Fever 326/396 (83%) 118/146 (81%) 0.687

Neck stiffness 325/421 (77%) 115/152 (76%) 0.528

Glasgow Coma Scale scoreb 11 (9-14) 10 (8-14) 0.039

Indices of cerebrospinal fluid inflammationc

Leukocyte count - cells/mm3 3232 (793-8675) 1700 (281-6538) 0.001

Glucose level – mmol/L 0.50 (0.00-2.60) 0.55 (0.20-2.21) <0.001

Protein level – g/L 0.15 (0.00-1.40) 4.18 (2.49-6.05) 0.046

Causative microorganism

S. pneumoniae 314 (72%) 150 (76 %) 0.867

N. meningitidis 63 (14%) 18 (9%) 0.363

Other 62 (14%) 29 (15%) 0.784

Mortality 35/435 (8%) 69/164 (42%) <0.001

Unfavorable outcome 114/435 (25%) 98/164 (60%) <0.001a Data are number/number evaluated (percentage), continuous data are median (interquartile range)

b Score on Glasgow Coma Scale was known in 434/439 (99%) patients with DNA and 162/197 (82%) patients without.c CSF leukocyte count was reported in 409/439 (93%) patients with DNA and 157/197 (80%) without, CSF glucose level was reported in 415/439 (95%) patients with DNA and 156/197 (79%) without, CSF protein level was reported in 412/439 (94%) patients with DNA and 154/197 (77%) without DNA.

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Supplementary Table 7.3. Allele frequency, Hardy-Weinberg equilibrium and genotyping success rate of evaluated common complement component polymorphisms in 287 white controls

HWEa Success

Gen SNP ID A % B % A B AA AB BB P - value rate

C3 rs1047286 78,6% 21,4% 451 123 179 93 15 0,816 99,4%

C3 rs2230199 77,1% 22,9% 438 130 169 100 15 0,999 98,9%

C5 rs17611 43,2% 56,8% 247 325 53 141 92 0,997 99,7%

C6 rs1801033 69,3% 30,7% 398 176 138 122 27 1,000 99,3%

C7 rs1063499 35,2% 64,8% 202 372 40 122 125 0,514 99,6%

C7 rs13157656 23,0% 77,0% 129 433 13 103 165 0,831 97,7%

C7 rs60714178 16,4% 83,6% 94 480 8 78 201 0,991 99,9%

C8B rs12067507 6,3% 93,7% 36 538 6 24 257 0,000 99,6%

C8B rs12085435 94,4% 5,6% 540 32 254 32 0 0,605 98,6%

C9 rs700233 61,7% 38,3% 343 213 106 131 41 0,999 95,7%

C9 rs34882957 94,3% 5,7% 532 32 250 32 0 0,600 98,7%

CFH rs505102 70,4% 29,6% 404 170 143 118 26 0,973 99,3%

CFH rs1065489 17,4% 82,6% 99 471 14 71 200 0,083 99,3%

CFH rs1410996 54,9% 45,1% 315 259 83 149 55 0,715 99,7%

CFH rs3753396 16,6% 83,4% 95 479 10 75 202 0,659 99,7%

CFH rs6677604 80,5% 19,5% 459 111 187 85 13 0,710 99,1%

CFH rs3753394 26,0% 74,0% 148 422 20 108 157 0,971 99,4%

aHardy Weinberg equilibrium.

Supplementary Table 7.4. Multivariate logistic regression analysis for unfavorable outcome in pneumococcal meningitis

Patient characteristic Odds ratio(95% confidence interval)

p- value

Age 1.017 (0.996 – 1.038) 0.107

Glasgow coma scale score 1.156 (1.279 – 1.045) 0.005

Thrombocyte count 1.000 (0.997 – 1.003) 0.976

CSF leukocyte count <1000/mm3 3.717 (2.058 – 6.711) <0.001

Rs17611 2.041 (1.144 – 3.636) 0.016

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Supplementary Figure 7.1. Effect of neutropenia in experimental pneumococcal meningitis

In order to asses the role of neutrophils, wild type mice were treated with 250 μg of either anti-GR1-antibody or rat IgG2b isotype control antibody (n=8 per group) 24 hours before disease induction. Then, animals were infected with S. pneumoniae and evaluated 24 h later for blood neutrophil counts, CSF leukocyte counts (CSF WBC count), intracranial pressure (ICP), blood brain barrier-breaching and intracerebral hemorrhage combined in the neuroscore. Anti-GR1-treatment resulted in markedly lower blood neutrophil and CSF leukocyte numbers compared to isotype control-treated mice which was also paralleled by a significant reduction in ICP and neuroscore values (unpaired Student’s test; data are shown as mean±SD).

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Supplementary Figure 7.2. Role of the membrane attack complex (MAC) in the mouse model of pneumococcal meningitis

To evaluate the role of MAC, mice deficient in C6 (C6-/-, n=14) and thus unable to form MAC and mice deficient in CD59 (Cd59a-/-, n=11), the in vivo inhibitor of MAC, were examined. Animals were infected with S. pneumoniae and evaluated at 24 h after infection for CSF leukocyte count (CSF WBC count), clinical score, intracranial pressure (ICP), blood brain barrier-breaching and intracerebral hemorrhage combined in the neuroscore and expression of proinflammatory mediators and cytokines, namely Interleukin-6 and MIP-2. Infected mouse mutants were compared to infected wt mice (C57BL/6 (BL6), f = female, n=20). C57BL/6 mice intracisternally injected with PBS served as controls (BL6 controls, n=8). Compared to infected wt mice and infected C6-/- mice, Cd59a-/- had significantly increased CSF WBC count. There was no difference in any of the other evaluated parameters (unpaired Student’s test; data are shown as mean±SD).

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Supplementary Figure 7.3. Role of C3a in the mouse model of pneumococcal meningitis

To evaluate the role of C3a, mice deficient in the C3a-receptor (C3ar1-/-, n=12) and mice with selective expression of C3a in the CNS (C3a/GFAP, n = 11) were examined. Animals were infected with S. pneumoniae and evaluated at 24 h after infection for CSF leukocyte count (CSF WBC count), clinical score, intracranial pressure (ICP), blood brain barrier-breaching and intracerebral hemorrhage combined in the neuroscore and expression of proinflammatory mediators and cytokines, namely Interleukin-6 and MIP-2. Infected mouse mutants were compared to infected wt mice (C57BL/6 (BL6), m = male, n=12). C57BL/6 mice intracisternally injected with PBS served as controls (BL6 controls, n=8). Compared to infected wt mice and infected C3ar1-/- mice, C3/GFAP mice had significantly increased CSF WBC count. There was no difference in any of the other of the evaluated parameters (unpaired Student’s test; data are shown as mean±SD).

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Supplementary methods

Participating hospitals, local investigators (number of patients included)

Academisch Medisch Centrum (number of patients included, 25), Amphia

Ziekenhuis, R.J. de Graaf (23), Universitair Medisch Centrum Sint Radboud, R.A.J.

Esselink (22), Atrium Medisch Centrum, M.J. Wennekes (18), Ziekenhuisgroep

Twente, J.C. Baart (18), Gelre Ziekenhuis, H.P. Bienfait, (18), Leids Universitair

Medisch Centrum, C.S.M. Straathof (15), Groene-Hart ziekenuis, G.A.M. Verheul

(15), Haga Ziekenhuis, R.W.M. Keunen (15), Universitair Medisch Centrum

Groningen, R.H. Enting (13), Medisch Centrum Alkmaar, R. ten Houten (13),

Meander Medisch Centrum, W.G.H. Oerlemans (13), Rijnstate Ziekenhuis, E.M.

Hoogerwaard (13), Tweesteden Ziekenhuis, J.P.L. van der Plas (13), Viecuri

Ziekenhuis, P.H.M. Pop (13), Slingeland Ziekenhuis, C.J.W. van de Vlasakker

(12), Tergooi Ziekenhuizen, M. Stevens, D. Herderschee (12), Westfries Gasthuis,

D. Broere (11), Catharina Ziekenhuis, J.N. Berendes (11), Beatrix Ziekenhuis,

R.B. Alting van Geusau (10), Isala Klinieken, J.S.P. van den Berg (10), Rijnland

Ziekenhuis, R.J.W. Witteveen (10), Sint Jansdal Ziekenhuis, T.F.M. Fennis (10),

Deventer ziekenhuizen, H.J.M.M. Lohman (9), Diakonessenhuis Utrecht, M.H.

Christiaans (9), Koningin Beatrix Ziekenhuis, R.C.F. Smits (9), Medisch Spectrum

Twente, J.A.G. Geelen (9), Boven-IJ Ziekenhuis, M.A. Struys (8), Gelderse Vallei

Ziekenhuis, C. Jansen (8), Jeroen Bosch Ziekenhuis, H.F. Visee (8), Orbis Medical

Concern, H.W.M. Anten (8), OLVG, I.N. van Schaik (8), Sint Elisabeth Ziekenhuis,

G.F.J. Brekelmans (8), StreekZiekenhuis Midden-twente, J.J.W. Prick (8), Albert

Schweitzer Ziekenhuis, H. Kerkhoff (7), Erasmus Medisch Centrum, B.C. Jacobs

(7), Kennemer Gasthuis, M. Weisfelt (7), Scheper Ziekenhuis, E.V. van Zuilen (7),

Ziekenhuis Zevenaar, A.van de Steen (7), Flevo Ziekenhuis, J.P. Blankevoort (6),

Elkerliek Ziekenhuis, A.J.M.Kok (6), Maasstadziekenhuis, R. Saxena (6), Hofpoort

Ziekenhuis, E.J. Wieringa (6), Rivierenland Ziekenhuis, P.J. de Jong (6), Zaans

Medisch Centrum, A. Koppenaal (6), Ziekenhuis Bernhoven, P.R. Schiphof

(5), Medisch Centrum Leeuwarden, W. van der Kamp (5), Reinier de Graaf

Ziekenhuis, W.J.H.M. Grosveld (5), VU Medisch Centrum, J.C. Reijneveld (5), Sint

Lucas Andreas Ziekenhuis, E.J. Wouda (5), Vlietland Ziekenhuis, C.J. Gijsbers

(5), Sint Franciscus Ziekenhuis, C. Bülens (4), Ziekenhuis de Lievensberg,

P.J.I.M. Berntsen (4), Slotervaart Ziekenhuis, I.H. Kwa (4), Sint Jansgasthuis,

R.H.J. Medaer (4), Antonius Ziekenhuis, R.S. Holscher (4), Bethesda Ziekenhuis,

J.P. Schipper (4), Canisius-Wilhelmina Ziekenhuis, G.W. van Dijk (4), Medisch

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Centrum Haaglanden, M.J.B. Taphoorn (4), Dirksland Ziekenhuis, U.W. Huisman

(4), Franciscus Ziekenhuis, A. van Spreeken (4), Gemini Ziekenhuis, P. Admiraal

(4), Sint Anna Ziekenhuis, H.B.M. van Lieshout (4), Sint Lucas Ziekenhuis,

A.N. Zorgdrager (4), Sint Laurentius Ziekenhuis, P.H.M.F. van Domburg (3),

Academisch Ziekenhuis Maastricht, Dr. E.P.M. van Raak (3), Bronovo Ziekenhuis,

M. Gerrits (3), IJsselmeerziekenhuizen, E.M. Leenders (3), Maasziekenhuis,

R.M.J.A.Roebroek (3), Martini Ziekenhuis Groningen, J.W. Snoek (3), Maxima

Medisch Centrum, A.J. Vermeij (3), Mesos Medisch Centrum, P.H. Wessels (3),

Oosterschelde Ziekenhuis, A.M. Boon (3), Refaja Ziekenhuis, L. Vrooland (3),

Röpcke-Zweers Ziekenhuis, J.G.M. Knibbeler (3), Ruwaard van Putten Ziekenhuis,

H.W. ter Spill (3), Spaarne Ziekenhuis, R.J. Meijer (3), Ziekenhuis De Sionsberg,

J.P. Krooman (2), IJsselland Ziekenhuis, J. Heerema (2), Waterland Ziekenhuis,

J.G.W. Oonk (2), Ziekenhuis Amstelland, D.S.M. Molenaar (2), Ziekenhuis

Walcheren, J.P. Koeman (2), Ziekenhuis Zeeuws-Vlaanderen, W. Hoefnagels (2),

Ziekenhuis de Tjongerschans, R.F. Duyff (2), Ziekenhuis Delfzicht, J.A. Don (1),

Diaconessenhuis Meppel, E.J.V. Keuter (1), Havenziekenhuis, R.J.W. Dunnewold

(1), Ziekenhuis Nij Smellinghe, K.D. Beintema (1), Rode Kruis Ziekenhuis, L.

Zegerius (1), Sint Antonius Ziekenhuis, H.W. Mauser (1), Wilhelmina Ziekenhuis,

A.E. Bollen (1).

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4. Tauber MG, Khayam-Bashi H, Sande MA. Effects of ampicillin and corticosteroids on brain water content, cerebrospinal fluid pressure, and cerebrospinal fluid lactate levels in experimental pneumococcal meningitis. J Infect Dis 1985;151(3):528-534.


6. van de Beek D, de Gans J. Dexamethasone in adults with community-acquired bacterial meningitis. Drugs 2006;66(4):415-427.



9. Brouwer MC, Heckenberg SG, de Gans J, Spanjaard L, Reitsma JB, van de Beek D. Nationwide implementation of adjunctive dexamethasone therapy for pneumococcal meningitis. Neurology 2010;75(10):1-7.

10. Hamburg MA, Collins FS. The path to personalized medicine. N Engl J Med 2010;363(4):301-304.

11. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2009;9(1):31-44.

12. Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 2010;11(9):785-797.

13. Jonsson G, Truedsson L, Sturfelt G, Oxelius VA, Braconier JH, Sjoholm AG. Hereditary C2 deficiency in Sweden: frequent occurrence of invasive infection, atherosclerosis, and rheumatic disease. Medicine (Baltimore) 2005;84(1):23-34.

14. Sprong T, Roos D, Weemaes C et al. Deficient alternative complement pathway activation due to factor D deficiency by 2 novel mutations in the complement factor D gene in a family with meningococcal infections. Blood 2006;107(12):4865-4870.

15. Biesma DH, Hannema AJ, van Velzen-Blad H et al. A family with complement factor D deficiency. J Clin Invest 2001;108(2):233-240.

16. Fijen CA, van den BR, Schipper M et al. Properdin deficiency: molecular basis and disease association. Mol Immunol 1999;36(13-14):863-867.

17. Haralambous E, Dolly SO, Hibberd ML et al. Factor H, a regulator of complement activity, is a major determinant of meningococcal disease susceptibility in UK Caucasian patients. Scand J Infect Dis 2006;38(9):764-771.

18. Davila S, Wright VJ, Khor CC et al. Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease. Nat Genet 2010;42(9):772-776.

19. Tuomanen E, Hengstler B, Zak O, Tomasz A. The role of complement in inflammation during experimental pneumococcal meningitis. Microb Pathog 1986;1(1):15-32.

20. Paul R, Obermaier B, Van ZJ et al. Myeloid Src kinases regulate phagocytosis and oxidative burst in pneumococcal meningitis by activating NADPH oxidase. J Leukoc Biol 2008;84(4):1141-1150.

21. Rupprecht TA, Angele B, Klein M et al. Complement C1q and C3 are critical for the innate immune response to Streptococcus pneumoniae in the central nervous system. J Immunol 2007;178(3):1861-1869.

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22. Gardner P. Clinical practice. Prevention of meningococcal disease. N Engl J Med 2006;355(14):1466-1473.

23. Jennett B, Teasdale G, Braakman R, Minderhoud J, Knill-Jones R. Predicting outcome in individual patients after severe head injury. Lancet 1976;1(7968):1031-1034.


25. Koedel U, Frankenberg T, Kirschnek S et al. Apoptosis is essential for neutrophil functional shutdown and determines tissue damage in experimental pneumococcal meningitis. PLoS Pathog 2009;5(5):e1000461.

26. Brown JS, Hussell T, Gilliland SM et al. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice. Proc Natl Acad Sci U S A 2002;99(26):16969-16974.

27. Koedel U, Klein M, Pfister HW. New understandings on the pathophysiology of bacterial meningitis. Curr Opin Infect Dis 2010;23(3):217-223.

28. Boos L, Campbell IL, Ames R, Wetsel RA, Barnum SR. Deletion of the complement anaphylatoxin C3a receptor attenuates, whereas ectopic expression of C3a in the brain exacerbates, experimental autoimmune encephalomyelitis. J Immunol 2004;173(7):4708-4714.

29. Klein M, Obermaier B, Angele B et al. Innate immunity to pneumococcal infection of the central nervous system depends on toll-like receptor (TLR) 2 and TLR4. J Infect Dis 2008;198(7):1028-1036.


31. Armstrong PW, Granger CB, Adams PX et al. Pexelizumab for acute ST-elevation myocardial infarction in patients undergoing primary percutaneous coronary intervention: a randomized controlled trial. JAMA 2007;297(1):43-51.

32. Hillmen P, Young NS, Schubert J et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med 2006;355(12):1233-1243.

33. Weisfelt M, van de Beek D, Spanjaard L, Reitsma JB, de Gans J. Attenuated cerebrospinal fluid leukocyte count and sepsis in adults with pneumococcal meningitis: a prospective cohort study. BMC Infect Dis 2006;6:149.

34. Giampaolo C, Scheld M, Boyd J, Savory J, Sande M, Wills M. Leukocyte and bacterial interrelationships in experimental meningitis. Ann Neurol 1981;9(4):328-333.

35. Tauber MG, Kennedy SL, Tureen JH, Lowenstein DH. Experimental pneumococcal meningitis causes central nervous system pathology without inducing the 72-kd heat shock protein. Am J Pathol 1992;141(1):53-60.

36. Tortajada A, Montes T, Martinez-Barricarte R, Morgan BP, Harris CL, de Cordoba SR. The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity. Hum Mol Genet 2009;18(18):3452-3461.

37. Goicoechea de JE, Harris CL, Esparza-Gordillo J et al. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci U S A 2007;104(1):240-245.

38. Hillebrandt S, Wasmuth HE, Weiskirchen R et al. Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nat Genet 2005;37(8):835-843.

39. Gressner O, Meier U, Hillebrandt S et al. Gc-globulin concentrations and C5 haplotype-tagging polymorphisms contribute to variations in serum activity of complement factor C5. Clin Biochem 2007;40(11):771-775.

40. Ernst JD, Hartiala KT, Goldstein IM, Sande MA. Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis. Infect Immun 1984;46(1):81-86.

41. Kadurugamuwa JL, Hengstler B, Bray MA, Zak O. Inhibition of complement-factor-5a-induced inflammatory reactions by prostaglandin E2 in experimental meningitis. J Infect Dis 1989;160(4):715-719.

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42. Tuomanen EI, Saukkonen K, Sande S, Cioffe C, Wright SD. Reduction of inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes. J Exp Med 1989;170(3):959-969.

43. Ritis K, Doumas M, Mastellos D et al. A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways. J Immunol 2006;177(7):4794-4802.

44. Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD. Complement and coagulation: strangers or partners in crime? Trends Immunol 2007;28(4):184-192.

45. Leung LL, Myles T, Nishimura T, Song JJ, Robinson WH. Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI). Mol Immunol 2008;45(16):4080-4083.

46. Vergouwen MD, Schut ES, Troost D, van de Beek D. Diffuse cerebral intravascular coagulation and cerebral infarction in pneumococcal meningitis. Neurocrit Care 2010;13(2):217-227.

47. Kastenbauer S, Pfister HW. Pneumococcal meningitis in adults: spectrum of complications and prognostic factors in a series of 87 cases. Brain 2003;126(Pt 5):1015-1025.

48. Jacob A, Hack B, Chiang E, Garcia JG, Quigg RJ, Alexander JJ. C5a alters blood-brain barrier integrity in experimental lupus. FASEB J 2010;24(6):1682-1688.

49. Liu ZM, Zhu SM, Qin XJ et al. Silencing of C5a receptor gene with siRNA for protection from Gram-negative bacterial lipopolysaccharide-induced vascular permeability. Mol Immunol 2010;47(6):1325-1333.

50. Farkas I, Baranyi L, Liposits ZS, Yamamoto T, Okada H. Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells. Neuroscience 1998;86(3):903-911.

51. Yanamadala V, Friedlander RM. Complement in neuroprotection and neurodegeneration. Trends Mol Med 2010;16(2):69-76.

52. Sprong T, Brandtzaeg P, Fung M et al. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. Blood 2003;102(10):3702-3710.

53. Brouwer MC, Read RC, van de Beek D. Host genetics and outcome in meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2010;10(4):262-274.

54. Woodruff TM, Strachan AJ, Dryburgh N et al. Antiarthritic activity of an orally active C5a receptor antagonist against antigen-induced monarticular arthritis in the rat. Arthritis Rheum 2002;46(9):2476-2485.

55. Vergunst CE, Gerlag DM, Dinant H et al. Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation. Rheumatology (Oxford) 2007;46(12):1773-1778.

56. Osmers I, Szalai AJ, Tenner AJ, Barnum SR. Complement in BuB/BnJ mice revisited: serum C3 levels and complement opsonic activity are not elevated. Mol Immunol 2006;43(10):1722-1725.

57. Malipiero U, Koedel U, Pfister HW et al. TGFbeta receptor II gene deletion in leucocytes prevents cerebral vasculitis in bacterial meningitis. Brain 2006;129(Pt 9):2404-2415.

58. Kastenbauer S, Koedel U, Becker BF, Pfister HW. Oxidative stress in bacterial meningitis in humans. Neurology 2002;58(2):186-191.

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General discussion

Bacterial meningitis:

epidemiology, pathophysiology and treatment

Adapted from: Bacterial meningitis: epidemiology, pathophysiology and

treatment. Sebastiaan G.B. Heckenberg, Matthijs C. Brouwer, D van de Beek

Handbook of Clinical Neurology 2013 (in press).

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Introduction

Community-acquired bacterial meningitis exacts a heavy toll, even in developed

countries. It is a neurological emergency and these patients require immediate

evaluation and treatment. The incidence of bacterial meningitis is about 5 cases

per 100,000 adults per year in developed countries and may be 10 times higher

in less developed countries.1, 2 The predominant causative pathogens in adults

are Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis

(meningococcus) which are responsible for about 80% of all cases.1, 2

Epidemiology

The incidence of acute bacterial meningitis is 5–10/100,000 persons per year in

high income countries, resulting in 15,000–25,000 cases in the USA annually.1,

3, 4 Vaccination strategies have substantially changed the epidemiology of

community-acquired bacterial meningitis during the past two decades.1, 3,

5 The routine vaccination of children against Haemophilus influenzae type B

has virtually eradicated H. influenzae meningitis in the developed world.1, 6

As a consequence, S. pneumoniae has become the most common pathogen

beyond the neonatal period and bacterial meningitis has become a disease

predominantly of adults. The introduction of conjugate vaccines against seven

serotypes of S. pneumoniae that are among the most prevalent in children aged

6 months to 2 years has reduced the rate of invasive pneumococcal infections

in young children and in older persons.5 The integration of the meningococcal

protein–polysaccharide conjugate vaccines into vaccination programs in

several countries further reduced the disease burden of bacterial meningitis in

high- and medium-income countries.7

S. pneumoniae affects all ages and causes the most severe disease in the very

young and the very old.8 Of the >90 pneumococcal serotypes, a few dominate

as the causes of meningitis. The increase of drug-resistant strains of S.

pneumoniae, is an emerging problem worldwide. The prevalence of antibiotic-

resistant strains in some parts of the US is as high as 50–70% with important

consequences for treatment.9

N. meningitidis is mainly responsible for bacterial meningitis in young adults;

it causes sporadic cases and epidemics.10 Its incidence shows a peak in winter

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and early spring and varies greatly around the world. Small outbreaks typically

occur in young adults living in close quarters, such as dormitories of military

camps or schools. Major epidemics have occurred periodically in sub-Saharan

Africa (the so-called ‘meningitis belt’), Europe, Asia and South America.1 During

these epidemics, attack rates can reach several hundred per 100,000, with

devastating consequences.

Meningococcal serogroups are determined by structural differences in

the capsular polysaccharide. Serogroups A, B, C, X, Y and W-135 are most

commonly associated with invasive disease worldwide, with regional variation

in the distribution.10 In the Netherlands, serogroup B and C account for >95%

of disease.chapter 2 Serogroup A meningococci cause epidemic disease in the

‘meningitis belt’ every 5-10 years, emphasising the importance of vaccination

in these parts of Africa.10 Multilocus sequence typing (MLST) has provided

an additional typing method based on the sequencing of 6 meningococcal

housekeeping genes.11 This unambiguous method allows for international

monitoring of meningococcal epidemiology through an online database

(pubmlst.org). In chapter 2, we described the relation of meningococcal clonal

complex with clinical characteristics. Meningococcal meningitis caused by

meningococci belonging to cc11 was associated with sepsis and poor outcome.

Clonal complex 11 was strongly associated with serogroup C and since our

study, the implementation of serogroup C vaccination in the Netherlands has

all but eradicated serogroup C disease. As serogroup B disease also reduced,

the share of meningococcal meningitis in all adult bacterial meningitis has

decreased from 37% (1998-2002) to 14% (2006-2009).chapter 5

The group B streptococcus (S. agalactiae) is a pathogen of neonates and often

causes a devastating sepsis and meningitis.1, 12 It colonizes the maternal birth

canal, and is transmitted to the child during delivery. The colonized newborn

can develop group B streptococcal disease of early onset (developing at less

than 7 days of age; median 1 day) or late onset (developing later than 7 days

of age). Listeria monocytogenes causes meningitis preferentially in neonates, in

adults with alcoholism, immunosupression, or iron overload, pregnant women

or the elderly.13 There is often an encephalitic component to presentation, with

early mental status alterations, neurologic deficits and seizures. In countries

with routine vaccination against H. influenzae type B it has become a rare

disease.6 In large parts of the world H. influenzae type b remains a major cause

of paediatric meningitis, with high rates of mortality and hearing loss.1

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Bacterial meningitis also occurs in hospitalized patients (“physician associated

meningitis” or “nosocomial meningitis”). In a large city hospital, almost 40%

of cases may be nosocomial.4 Most cases occur in patients undergoing

neurosurgical procedures, including implanting of neurosurgical devices,

and in patients with focal infections of the head. Furthermore, patients with

cerebrospinal fluid shunts are at continuous risk of developing drain associated

meningitis.14 The organisms causing nosocomial meningitis differ markedly

from those causing community-acquired meningitis and include Gram-

negative rods (e.g. Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa,

Acinetobacter spp., Enterobacter spp.), staphylococci and streptococci other

than S. pneumoniae.14-16

Since the early antibiotic era, the emergence of antimicrobial resistance has

been a continuing problem. Pneumococcal resistance to penicillin, due to

changes in its penicillin binding proteins, started to appear in the 1960s and

has since developed worldwide, often necessitating initial therapy with a

combination of a third-generation cephalosporin with vancomycin, instead of

monotherapy with penicillin.1

Genetics

Host genetic factors are major determinants of susceptibility to infectious

diseases. A cause of these differences in susceptibility are single base-pair

variations, also known as single-nucleotide polymorphisms (SNPs), in genes

controlling the host response to microbes. Patients with recurrent or familial

meningitis or sepsis due to S. pneumoniae or N. meningitidis are often found

to have rare mutations that cause a substantial increase in susceptibility

to infection.17 These mutations are mostly founding genes coding for the

complement system or Toll like receptor (TLR) pathways. In the general

population identified alterations include SNPs in the complement system,

cytokines and TLRs.17 A genome wide association study has shown complement

factor H SNPs decrease the risk of meningococcal disease.18 In patients with

sepsis due to N. meningitidis, SNPs in cytokine and fibrinolysis genes have been

reported to influence mortality.19

In bacterial meningitis research on genetic factors is lacking but may provide

important pathophysiological insights. Bacterial meningitis is a complex

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disorder in which injury is caused, in part, by the causative organism and, in

part, by the host’s own inflammatory response. Recognition of particular

subgroups of patients with a genetic predisposition to more severe illness may

help to individualize treatment and improve prognosis.

We performed a nationwide genetic association study in adults with bacterial

meningitis on common variants in the complement system and selected

all SNPs with a minor allele frequency of more than 5% in genes coding for

complement components (C1QA, C1QB, C1QC, C2, C3, C5, C6, C7, C8B, C9, CFD,

CFH, CFI, and CFP) for which a commercial genotyping assay was available.

We identified rs17611 in complement component 5 (C5; GG genotype) to be

associated with unfavorable outcome in patients of mixed European descent

with pneumococcal meningitis (OR, 2.25; 95% CI, 1.33–3.81; p=0.002). chapter 7

Pathophysiology and pathology

Specific bacterial virulence factors for meningeal pathogens include specialized

surface components that are crucial for adherence to the nasopharyngeal

epithelium, the evasion of local host defense mechanisms and subsequent

invasion of the bloodstream.20 In pneumococcal disease, presence of the

polymeric immunoglobulin A receptor on human mucosa, which binds to a

major pneumococcal adhesin, CbpA, correlates with the ability of pneumococci

to invade the mucosal barrier. Viral infection of the respiratory tract may also

promote invasive disease.21 From the nasopharyngeal surface, encapsulated

organisms cross the epithelial cell layer and invade the small subepithelial

blood vessels.

Binding of bacteria to upregulated receptors (e.g., platelet activating-factor

receptors) promotes migration through the respiratory epithelium and vascular

endothelium, resulting in blood stream invasion.

In the bloodstream, bacteria must survive host defenses, including circulating

antibodies, complement-mediated bactericidal mechanisms and neutrophil

phagocytosis. Encapsulation is a shared feature of the principal meningeal

pathogens. To survive the various host conditions they encounter during

infection, pneumococci undergo spontaneous and reversible phase variation,

which involves changes in the amount of important surface components.21,

22 The capsule is instrumental in inhibiting neutrophil phagocytosis and

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complement-mediated bactericidal activity. Several defense mechanisms

counteract the antiphagocytic activity of the bacterial capsule. Activation of

the alternative complement pathway results in cleavage of C3 with subsequent

deposition of C3b on the bacterial surface, thereby facilitating opsonization,

phagocytosis and intravascular clearance of the organism.23 Impairment of the

alternative complement pathway occurs in patients with sickle-cell disease

and those who have undergone splenectomy, and these groups of patients

are predisposed to the development of pneumococcal meningitis. Functional

deficiencies of several components involved in the activation and function of

complement-mediated defences have been identified (i.e., mannose-binding

lectin, properdin, terminal complement components), which increase the

susceptibility for invasive meningococcal infections.17 In our studies following

the discovery of the role of the rs17611-SNP in pneumococcal meningitis,

the anaphylatoxin C5a was identified as a crucial complement product in

pneumococcal meningitis. Neutralization experiments showed that adjunctive

treatment with C5-Ab improved outcome in mice with pneumococcal

meningitis. chapter 7

The blood–brain barrier is formed by cerebromicrovascular endothelial cells,

which restrict blood-borne pathogen invasion. Cerebral capillaries, as opposed

to other systemic capillaries, have adjacent endothelial cells fused together

by tight junctions that prevent intercellular transport.24 Bacteria are thought

to invade the subarachnoid space via transcytosis. Nonhaematogenous

invasion of the CSF by bacteria occurs in situations of compromised integrity

of the barriers surrounding the brain. Direct communication between the

subarachnoid space and the skin or mucosal surfaces as a result of malformation

or trauma gives rise to meningeal infection. Bacteria can also reach the CSF as

a complication of neurosurgery or spinal anesthesia.14

Physiologically, concentrations of leucocytes, antibodies, and complement

components in the subarachnoid space are low, which facilitates rapid

multiplication of bacteria. In the CSF pneumococcal cell-wall products,

pneumolysin, and bacterial DNA induce a severe inflammatory response via

binding to Toll-like receptor-2 (TLR).24 Once engaged, this signaling receptor

transmits the activating signal into the cell, which initiates the induction of

inflammatory cytokines.25 In N. meningitidis, lipopolysaccharide (LPS) is a

major component of the outer membrane. LPS is sensed by mammalian cells

through Toll-like receptor 4 (TLR4), in combination with coreceptors MD-2 and

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CD14. The host responds to bacterial endotoxin with proinflammatory gene

expression and activation of coagulation pathways in sepsis and meningitis.10

The discovery of meningococcal lpxL1 mutants and the associated reduced

induction of pro-inflammatory cytokines prompted our investigation of

the clinical fenotype caused by meningococcal lpxL1 mutants in adults with

meningococcal meningitis. chapter 3 Infection with lpxL1-mutant meningococcal

strains is associated with less systemic inflammation and reduced activation of

the coagulant system, reflected in less fever, higher serum platelet counts, and

lower numbers with rash.

The subarachnoid inflammatory response is accompanied by production of

multiple mediators in the CNS. Tumour necrosis factor α (TNFα), interleukin

1β, and interleukin 6 are regarded as the major early response cytokines that

trigger the inflammatory cascade, which induces various pathophysiological

alterations implicated in pneumococcal meningitis (Figure 1).20 TNFα and

interleukin 1β stimulate the expression of chemokines and adhesion molecules,

which play an important part in the influx of leucocytes from the circulation

to the CSF. Upon stimulation with bacterial components, macrophages and

granulocytes release a broad range of potentially tissue-destructive agents,

which contribute to vasospasm and vasculitis, including oxidants (e.g.,

peroxynitrite) and proteolytic enzymes such as matrix metalloproteinases

(MMP). Matrix metalloproteinases (MMP), zinc-dependent enzymes produced

as part of the immune response to bacteria that degrade extracellular matrix

proteins, also contribute to the increased permeability of the blood–brain

barrier.

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Figure 1. Multiple complications in a patient with pneumococcal meningitis

(A) T2-proton-density-weighted MRI of the brain shows a transverse view of a hyperintense signal (arrows) in the globus pallidus, putamen and thalamus that indicates bilateral edema. (B) A postmortem view of the brain of the same patient shows yellowish-colored meninges as a result of extensive inflammation. (C) Confirmation of the bilateral infarction of globus pallidus, putamen and thalamus (arrows). The microscopic substrate in the same patient shows a meningeal artery with (D) lymphocytic infiltration in and around the vessel wall, (E) extensive subpial necrotizing cortical inflammation, and (F) edema in the white matter.

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A major contributor to increased intracranial pressure in bacterial meningitis

is the development of cerebral oedema, which may be vasogenic, cytotoxic or

interstitial in origin. Vasogenic cerebral oedema is a consequence of increased

blood–brain barrier permeability.26 Cytotoxic edema results from an increase

in intracellular water following alterations of the cell membrane and loss of

cellular homeostasis. Cytotoxic mechanisms include ischemia and the effect of

excitatory amino acids. Secretion of antidiuretic hormone also contributes to

cytotoxic oedema by making the extracellular fluid hypotonic and increasing

the permeability of the brain to water. Interstitial oedema occurs by an increase

in CSF volume, either through increased CSF production via increased blood

flow in the choroid plexus, or decreased resorption secondary to increased CSF

outflow resistance.

The exact mechanisms that lead to permanent brain injury are incompletely

understood. Cerebral ischemic necrosis probably contributes to damage to

the cerebral cortex (Figure 1). Cerebrovascular complications occur in 15–20%

of patients with bacterial meningitis.2 Other abnormalities include subdural

effusion or empyema, septic sinus thrombosis, subarachnoid hematomas,

compression of intracranial structures due to intracranial hypertension,

and herniation of the temporal lobes or cerebellum. Gross changes, such as

pressure coning, are rare.27

There is diffuse acute inflammation of the pia-arachnoid, with migration of

neutrophil leucocytes and exudation of fibrin into the CSF. Pus accumulates

over the surface of the brain, especially around its base and the emerging

cranial nerves, and around the spinal cord. The meningeal vessels are dilated

and congested and may be surrounded by pus (Figure 1). Pus and fibrin

are found in the ventricles and there is ventriculitis, with loss of ependymal

lining and subependymal gliosis. Infection may block CSF circulation, causing

obstructive hydrocephalus or spinal block. In many cases death may be

attributable to related septicaemia, although bilateral adrenal haemorrhage

(Waterhouse–Friederichsen syndrome) may well be a terminal phenomenon

rather than a cause of fatal adrenal insufficiency as was once imagined. Patients

with meningococcal septicaemia may develop acute pulmonary oedema.

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Clinical presentation

Community-acquired bacterial meningitis

Early diagnosis and rapid initiation of appropriate therapy are vital in the

treatment of patients with bacterial meningitis. A recent study provided a

systematic assessment of the sequence and development of early symptoms

in children and adolescents with meningococcal disease (encompassing

the spectrum of disease from sepsis to meningitis) before admission to the

hospital.28 Classic symptoms of rash, meningismus, and impaired consciousness

develop late in the pre-hospital illness, if at all. Early signs before admission

in adolescents with meningococcal disease were leg pain and cold hands and

feet.

Bacterial meningitis is often considered but may be difficult to recognize. The

clinical presentation of a patient with bacterial meningitis may vary depending

on age, underlying conditions and severity of illness. Clinical findings of

meningitis in young children are often minimal and in childhood bacterial

meningitis and in elderly patients’ classical symptoms such as headache, fever,

nuchal rigidity and altered mental status may be less common than in younger

and middle-aged adults.1, 29 Infants may become irritable or lethargic, stop

feeding, and are found to have a bulging fontanel, separation of the cranial

sutures, meningism, and opisthotonos, and they may develop convulsions.

These findings are uncommon in neonates, who sometimes present with

respiratory distress, diarrhoea, or jaundice.1, 12 In a prospective study on adults

with bacterial meningitis, the classic triad of signs and symptoms consisting

of fever, nuchal rigidity and altered mental status was present in only 44%

of the patients.30 Certain clinical features may predict the bacterial cause of

meningitis. Predisposing conditions like ear or sinus infections, pneumonia,

immunocompromise, and dural fistulae are estimated to be present in 68-92%

of adults with pneumococcal meningitis.8, 31 Rashes occur more frequently in

patients with meningococcal meningitis, with reported sensitivities of 63–80%

and with specificities of 83–92%.1, 32

Post-traumatic bacterial meningitis

This is often indistinguishable clinically from spontaneous meningitis.14, 16

However, in obtunded or unconscious patients who have suffered a recent or

previous head injury, few clinical signs may be present. A fever and deterioration

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in the level of consciousness or loss of vital functions may be the only signs of

meningitis. Finding a CSF leak adds support to the possibility of meningitis in

such patients, but this is undetectable in most cases. The range of bacteria

causing meningitis in these patients is broad and consideration should be

given to broad spectrum antibiotics including metronidazole for anaerobic

pathogens.

Infections of CSF shunts

Patients may present with clinical features typical of spontaneous meningitis,

especially if virulent organisms are involved.14 The more usual presentation is

insidious, with features of shunt blockage such as headache, vomiting, fever,

and a decreasing level of consciousness. Fever is a helpful sign, but is not a

constant feature and may be present in as few as 20 per cent of cases. Shunts

can be infected without causing meningitis, in which event the features

of the infection will be determined by where the shunt drains. Infection of

shunts draining into the venous system produces a disease similar to chronic

right-sided infective endocarditis together with glomerulonephritis (shunt

nephritis), while infection of shunts draining into the peritoneal cavity produces

peritonitis.

Management

Given the high mortality of acute bacterial meningitis, starting treatment

and completing the diagnostic process should be carried out simultaneously

in most cases (Figure 2).2 The first step is to evaluate vital functions, obtain

two sets of blood cultures, and blood tests which typically should not take

more than one or two minutes. At the same time, the severity of the patient’s

condition and the level of suspicion for the presence of bacterial meningitis

should be determined.

Recommendations for cranial CT and fears of herniation are based on the

observed clinical deterioration of a few patients in the several to many hours

after lumbar puncture and the perceived temporal relationship of lumbar

puncture and herniation, but proving a cause and effect association is very

difficult based on the available data. Therefore, it is reasonable to proceed with

lumbar puncture without a CT scan if the patient does not meet any of the

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following: patients who have new-onset seizures (suggestive of focal brain

lesions), an immunocompromised state, signs suspicious for space-occupying

lesions (papilledema or focal neurological signs - not including cranial nerve

palsy), or moderate-to-severe impairment of consciousness (score on the

Glasgow Coma Scale below 11).2, 27 Other contraindications to lumbar puncture

include local skin sepsis at the site of puncture, a clinically instable patient, and

any clinical suspicion of spinal cord compression. Lumbar puncture may also

be harmful in patients with coagulopathy, because of the chance of needle-

induced subarachnoid hemorrhage or of the development of spinal subdural

and epidural hematomas. Contraindications for (immediate) lumbar puncture

are provided in Table 1.2 In patients with suspected bacterial meningitis who

receive a CT scan before lumbar puncture, initial therapy consisting of adjunctive

dexamethasone (10 mg iv) and empirical antimicrobial therapy (Figure 2, Table

1) should always be started without delay, even before sending the patient to

the CT scanner. Several studies have reported a strong increase in mortality due

to a delay in treatment caused by cranial imaging.33 In chapter 2 we described

adults with meningococcal meningitis and neuroimaging preceded lumbar

puncture in 85 of 92 (92%) patients; antibiotics were administered before CT in

only 15 of 88 (17%) of these patients. Therefore, 83% of these patients suffered

a delay of administration of adequate therapy.

Table 1. Contra-indications for immediate lumbar puncture

Signs suspect for space occupying lesion: • Papilledema• Focal neurologic signs (excluding isolated cranial nerve palsies)

Score on Glasgow Coma Scale <10

Severe immunodeficiency (such as HIV)

New onset seizures

Skin infection puncture site

Coagulopathy, e.g. use of anticoagulant medication or clinical signs of diffuse intravascular coagulation

Septic shock

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Figure 2. Algorithm for the management of the patient with suspected community-bacterial meningitis

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Laboratory diagnosis

When CSF analysis shows increased white blood cell counts, confirming

a diagnosis of meningitis,it is important to discriminate between the

usually harmless viral and the life-threatening bacterial meningitis. The CSF

abnormalities of bacterial meningitis include raised opening pressure in almost

all patients, polymorphonuclear leukocytosis, decreased glucose concentration,

and increased protein concentration. In bacterial meningitis, the white blood

cell count is typically >1000 cells/µl, while in viral meningitis it is <300 cells/

µl, although there is considerable overlap.1, 27 The neutrophil count is higher

in bacterial than in viral meningitis. More than 90% of cases present with CSF

white cell counts of more than 100/µl. In immunocompromised patients, CSF

white blood cell counts may be lower, although acellular CSF is exceedingly

rare, except in patients with tuberculous meningitis.34 The normal CSF glucose

concentration is between 2.5 and 4.4 mmol/l which is approximately 65% of the

serum glucose. In bacterial meningitis the glucose concentration is usually less

than 2.5 mmol/l, or < 40% of the serum glucose. The CSF protein in bacterial

meningitis is usually increased >50 mg/dl.1, 30

The CSF Gram stain identifies the cauasative micro-organism in 50–90% of

cases and CSF culture is positive in 80% of untreated patients, depending on

the pathogen.1, 30 Gram’s staining of CSF permits the rapid identification of the

causative organism (sensitivity, 60-90%; specificity, >97%). The yield of CSF

Gram staining is only marginally decreased if the patient received antibiotic

treatment prior to the lumbar puncture.1 Latex particle agglutination tests that

detect antigens of N. meningitidis, S. pneumoniae, H. influenzae and S. agalactiae

have been tested in bacterial meningitis patients. No incremental yield of this

method was observed in several cohort studies. Therefore these tests are no

longer advised.1 In the past decade PCR has proven to provide additional yield

in recognizing the causative pathogen in bacterial meningitis patients from

CSF. The reported sensitivities and specificities are high for different organisms

and therefore PCR can be used to detect patients in whom cultures remain

negative or those who were pre-treated with antitbiotics. However, CSF culture

will remain the “gold standard” for diagnosis as it is obligatory to obtain the

in vitro susceptibility of the causative microorganism and to rationalize

treatment.1

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When to repeat a lumbar puncture

A repeat analysis of the cerebrospinal fluid should only be carried out in

patients whose condition has not responded clinically after 48 hours of

appropriate antimicrobial and adjunctive dexamethasone treatment.30 It

is essential when pneumococcal meningitis caused by penicillin-resistant

or cephalosporin-resistant strains is suspected. Gram staining and culture

of the cerebrospinal fluid should be negative after 24 hours of appropriate

antimicrobial therapy.

Serum markers of inflammation

In the distinction between viral and bacterial meningitis, serum inflammatory

markers may suggest the diagnosis.1, 30 Retrospective studies showed that

increased serum procalcitonin levels (>0.5 ng/ml) and C-reactive protein levels

(>20 mg/liter) were associated with bacterial meningitis.35, 36 Although elevated

concentrations can be suggestive of bacterial infection, they do not establish

the diagnosis of bacterial meningitis.

Blood Culture

Blood cultures are valuable to detect the causative organism and establish

susceptibility patterns if CSF cultures are negative or unavailable. Blood culture

positivity differs for each causative organism and varies between 50 and 90%.

The yield of blood cultures is decreased by 20% for patients who received

pretreatment with antibiotics.1

Skin biopsy

Microbiological examination of skin lesions is routine diagnostic work-up

in patients with suspected meningococcal infection. It differentiates well

between meningitis with and without haemodynamic complications, and the

result is not affected by previous antibiotic treatment.37, 38

Antimicrobial therapy

The choice of initial antimicrobial therapy is based on the most common

bacteria causing the disease according to the patient’s age, the clinical setting

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and on patterns of antimicrobial susceptibility (Table 2). Once the pathogen

has been isolated, specific treatment based on the susceptibility of the isolate

can replace the empirical regimen (Table 3).

The pharmacokinetics and dynamics of antimicrobial agents are important

drug characteristics to base the empirical regimen on. Penetration of the

BBB into the subarachnoid space is the first pharmacological factor that

determines whether an antimicrobial agent is able to clear bacteria from the

CSF. BBB penetration is affected by lipophility, molecular weight and structure,

and protein-bound fraction. Bacterial meningitis is a dynamic process and

CSF penetration of antimicrobials is highly dependent on the breakdown of

the BBB. Anti-inflammatory drugs such as dexamethasone might influence

the breakdown of the BBB and thereby interfere with CSF penetration of

antimicrobial agents.

The activity of antimicrobial drugs in infected purulent CSF depends on

a number of factors, such as activity in the environment of decreased pH,

protein-bound fraction, bacterial growth rate and density, and clearance in

the CSF. Mechanisms of antibiotic action are targeting of the bacterial cell wall,

targeting of the bacterial cell membrane or targeting biosynthetic processes.

Whereas bacteriostatic activity involves inhibition of growth of microorganisms,

bactericidal antimicrobials cause bacterial cell death. Antibiotic-induced lysis of

bacteria leads to the release of immunostimulatory cell-wall components and

toxic bacterial products, which induce a severe inflammatory response that

mainly occurs through binding to Toll-like receptors and the complement system.

Neonatal meningitis is largely caused by group B streptococci, E. coli, and L.

monocytogenes. Initial treatment, therefore, should consist of penicillin or

ampicillin plus a third-generation cephalosporin, preferably cefotaxime or

ceftriaxone, or penicillin or ampicillin and an aminoglycoside.1, 26, 39

In the community, children are at risk of meningitis caused by N. meningitidis

and S. pneumoniae, and, rarely in Hib-vaccinated children, H. influenzae.

Antimicrobial resistance has emerged among the three major bacterial

pathogens causing meningitis. Although intermediate penicillin resistance

is common in some countries, the clinical importance of penicillin resistance

in the meningococcus has yet to be established. Because of the resistance

patterns of these bacteria third-generation cephalosporins cefotaxime or

ceftriaxone should be used in children. 1, 12, 26, 39

Spontaneous meningitis in adults is usually caused by S. pneumoniae or N.

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meningitidis. Due to the worldwide emergence of multidrug-resistant strains

of S. pneumoniae, some experts recommend to add vancomycin to the initial

empiric antimicrobial regimen in adult patients. 1, 26, 39 Additionally, in patients

aged over 50 years treatment with ampicillin should be added to the above

antibiotic regimen for additional coverage of L. monocytogenes, which is more

prevalent among this age group. Although no clinical data on the efficacy of

rifampin in patients with pneumococcal meningitis are currently available,

some experts would recommend the use of this agent in combination with a

third-generation cephalosporin, with or without vancomycin, in patients with

pneumococcal meningitis caused by bacterial strains that, on the basis of local

epidemiology, are likely to be highly resistant to penicillin or cephalosporin. S.

suis remains sensitive to the β-lactams and should be treated with penicillin,

cefotaxime, or ceftriaxone. Fluoroquinolones may be an alternative.

Nosocomial post-traumatic meningitis is mainly caused by multiresistant

hospital-acquired organisms such as K. pneumoniae, E. coli, Pseudomonas

aeruginosa, and S. aureus. Depending on the pattern of susceptibility in a

given hospital unit, ceftazidime (2 g intravenously, every 8 h), cefotaxime,

ceftriaxone, or meropenem should be chosen. If P. aeruginosa infection seems

likely, ceftazidime or meropenem is the preferred antibiotic.14, 39

Device- and shunt-associated meningitis is caused by a wide range of

organisms, including methicillin-resistant staphylococci (mostly coagulase-

negative staphylococci) and multiresistant aerobic bacilli. Cases with shunts

and an insidious onset are probably caused by organisms of low pathogenicity,

and empirical therapy is a less urgent requirement. For postoperative

meningitis the first-line empirical therapy should be cefotaxime, or ceftriaxone,

or meropenem. If the patient has received broad-spectrum antibiotics recently

or if P. aeruginosa is suspected, ceftazidime or meropenem should be given.

Meropenem should be used if an extended-spectrum, β-lactamase organism

is suspected, and flucloxacillin or vancomycin if S. aureus is likely. The infected

shunt or drain will almost certainly have to be removed urgently.14

Once the aetiological agent has been isolated and its susceptibilities determined,

the empirical treatment should be changed, if necessary, to an agent or agents

specific for the isolate (Table 4). The optimal duration of treatment has not been

determined by rigorous scientific investigation; however, treatment regimens

that are probably substantially in excess of the minimum necessary to achieve

cure have been based on wide clinical experience.

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General recommendations for empirical antibiotic treatment have included

ceftriaxone administered intravenously every 12 h or intravenous cefotaxime

every 4 to 6 h, and/or ampicillin at 4-h intervals, or penicillin G every 4 h. There

are no randomized comparative clinical studies of the various dosing regimens.

In general, 7 days of antimicrobial therapy are given for meningitis caused by N.

meningitidis and H. influenzae, 10 to 14 days for S. pneumoniae, and at least 21

days for L. monocytogenes. As these guidelines are not standardized it must be

emphasized that the duration of therapy may need to be individualized on the

basis of the patient’s response. 1, 26, 39

Table 2. Recommendations for empirical antimicrobial therapy in suspected bacterial meningitis

Predisposing factor Common bacterial pathogens Initial intravenous antibiotic therapy

Age

<1 month Streptococcus agalactiae, Escherichia coli, Listeria monocytogenes

Ampicillin plus cefotaxime or an aminoglycoside

1-3 months S. pneumoniae, Neisseria meningitidis, S. agalactiae, Haemophilus influenzae, E. coli, L. monocytogenes

Ampicillin plus vancomycine plus ceftriaxone or cefotaxime†

4-23 months S. pneumoniae, N. meningitidis, S. agalactiae, H. influenzae, E. coli

Vancomycine plus ceftriaxone or cefotaxime†

2-50 years N. meningitidis, S. pneumoniae Vancomycine plus ceftriaxone or cefotaxime†

>50 years N. meningitidis, S. pneumoniae, L. monocytogenes, aerobic gram-negative bacilli

Vancomycine plus ceftriaxone or cefotaxime plus ampicillin‡

With risk factor present¶

S. pneumoniae, L. monocytogenes, H. influenza

Vancomycine plus ceftriaxone or cefotaxime plus ampicillin

Posttraumatic S. pneumoniae, H. influenzae Vancomycine plus ceftriaxone or cefotaxime plus ampicillin

Postneurosurgery Coagulase-negative staphylococci, Staphylococcus aureus, aerobic gram-negative bacilli (including Pseudomonas aeruginosa)

Vancomycin plus ceftazidime

CSF shunt Coagulase-negative staphylococci, S. aureus, aerobic gram-negative bacilli (including Pseudomonas aeruginosa), Propionibacterium acnes

Vancomycin plus ceftazidime

†Footnote: †In areas with very low penicillin-resistance rates (as in such as the Netherlands) monotherapy penicillin may be considered. ‡In areas with very low penicillin-resistance and cephalosporin-resistance rates (as in such as the Netherlands) combination therapy of amoxicillin and third-generation cephalosporin may be considered. ¶Alcoholism, altered immune status. General recommendations for intravenous empirical antibiotic treatment have included penicillin, 2 million units every 4 hours; amoxicillin or ampicillin, 2 g every 4 hours; vancomycin, 15 mg/kg every 6-8 hours; third-generation cephalosporin: ceftriaxone, 2 g every 12 hours, or cefotaxime, 2 g every 4-6 hours; ceftazidime, 2 g every 8 hours. This material was published previously by 2 articles by van de Beek et al as part of an online supplementary appendix to reference 1 and 24. Copyright 2006 and 2010 Massachusetts Medical Society.2, 14 All rights reserved.

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Table 3. Recommendations for specific antimicrobial therapy in suspected bacterial meningitis

Micro-organism Antimicrobial therapy Duration of therapy

H. influenzae

β-lactamase negative Amoxicilline 7 days

β-lactamase positive Ceftriaxone or cefotaxime 7 days

N. meningitidis

Penicillin MIC <0.1 µg/ml Penicillin G or amoxicilline 7 days

Penicillin MIC 0.1–1.0 µg/ml Ceftriaxone or cefotaxime 7 days

S. pneumoniae

Penicillin MIC <0.1 µg/ml Penicillin G or amoxicilline 10-14 days

Penicillin MIC 0.1–1.0 µg/ml Ceftriaxone or cefotaxime 10-14 days

Penicillin MIC >2.0 µg/ml or cefotaxime / ceftriaxone MIC >1.0 mg/ml

Vancomycin plus ceftriaxone or cefotaxime

10-14 days

L. monocytogenes Penicillin G or amoxicilline >21 days

S. agalactiae Penicillin G or amoxicilline 7 days

Adjunctive dexamethasone treatment

Animal models of bacterial meningitis showed that bacterial lysis, induced

by antibiotic therapy, leads to inflammation in the subarachnoid space. The

severity of this inflammatory response is associated with outcome and can be

attenuated by treatment with steroids.40 On basis of experimental meningitis

studies, several clinical trials have been undertaken to determine the effects of

adjunctive steroids in children and adults with bacterial meningitis.41, 42

Of several corticosteroids, the use of dexamethasone in bacterial meningitis has

been investigated most extensively. Dexamethasone is a glucocorticosteroid

with anti-inflammatory as well as immunosuppressive properties and has

excellent penetration in the CSF. In a meta-analysis of randomized trials since

1988, adjunctive dexamethasone was shown to reduce meningitis-associated

hearing loss in children with meningitis due to H. influenzae type B.43

As most available studies on adjunctive dexamethasone therapy in adults with

bacterial meningitis were limited by methodological flaws, its value in adults

remained a subject of debate for a long time. In 2002, results of a European

randomized placebo-controlled trial showed that adjunctive treatment with

dexamethasone, given before or with the first dose of antimicrobial therapy,

was associated with a reduction in the risk of unfavorable outcome in adults

with bacterial meningitis (relative risk [RR] 0.59; 95 per cent confidence interval

[CI] 0.37-0.94) and with a reduction in mortality (RR 0.48; CI 0.24-0.96).44 This

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beneficial effect was most apparent in patients with pneumococcal meningitis,

in whom mortality was decreased from 34 to 14%. The benefits of adjunctive

dexamethasone therapy were not undermined by an increase of severe

neurological disability in patients who survived or by any corticosteroid-

induced complication. In a post-hoc analysis, which included only patients

with pneumococcal meningitis who died within 14 days after admission, the

mortality benefit of dexamethasone therapy was entirely due to reduced

mortality from systemic causes such as septic shock, pneumonia or acute

respiratory distress syndrome; there was no significant reduction in mortality

due to neurological causes.45

Results of a subsequent quantitative review on this topic in adults, which

included five clinical trials, confirmed that treatment with corticosteroids was

associated with a significant reduction in mortality (RR 0.6; CI 0.4-0.8) and in

neurological sequelae (RR 0.6; CI 0.4-1). The reduction in case fatality in patients

with pneumococcal meningitis was 21% (RR 0.5; CI 0.3-0.8).46 In meningococcal

meningitis, in which the number of events was smaller, there were favorable

point estimates for preventing mortality (RR 0.9; CI 0.3-2.1) and neurological

sequelae (RR 0.5; CI 0.1-1.7), but these effects did not reach statistical

significance. Adverse events were equally divided between the treatment and

placebo groups. Treatment with adjunctive dexamethasone did not worsen

long-term cognitive outcome in adults after bacterial meningitis.47 Since the

publication of these results, adjunctive dexamethasone has become routine

therapy in most adults with suspected bacterial meningitis.31

Randomized studies in adults with bacterial meningitis from Malawi and

Vietnam have been published.48, 49 In the Malawi study dexamethasone was not

associated with any significant benefit, 49 whilst in Vietnam a significant benefit

in mortality (RR 0.43, CI 0.2–0.94) was seen in patients with confirmed bacterial

meningitis only.48 A recent meta-analysis of individual patient data of 5 recent

randomized controlled trials showed no effect of adjunctive dexamethasone in

meningitis.50 Guidelines recommend routine use of adjunctive dexamethasone

in adults with pneumococcal meningitis in high-income countries.2, 39

Dexamethasone therapy has been implemented on a large scale as adjunctive

treatment of adults with pneumococcal meningitis in the Netherlands.31 The

prognosis of pneumococcal meningitis on a national level has substantially

improved after the introduction of adjunctive dexamethasone therapy with a

reduction in mortality from 30 to 20%.

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Despite these encouraging results, the use of adjunctive dexamethasone

in bacterial meningitis remains controversial in certain patients. Clearly the

results from studies in children and adults from Malawi suggests that there is

no benefit in patients in that setting compared with the results from Europe

and Vietnam and further work is needed to determine if these differences in

the efficacy of dexamethasone can be explained. Secondly, patients with

septic shock and adrenal insufficiency benefit from corticosteroid therapy in

physiological doses and for >4 days; however, when there is no evidence of

relative adrenal insufficiency, therapy with corticosteroids may be detrimental.

Results of a subsequent quantitative review on this topic that included nine

studies comparing mortality rates of corticosteroid treatment in sepsis or

septic shock showed a trend towards increased mortality associated with their

administration.51 As controlled studies of the effects of corticosteroid therapy in

a substantial number of patients with both meningitis and septic shock are not

available at present, treatment with corticosteroids cannot be recommended

unequivocally for such patients. Third, corticosteroids may potentiate ischemic

and apoptotic injury to neurons. In animal studies of bacterial meningitis

corticosteroids aggravated hippocampal neuronal apoptosis and learning

deficiencies in dosages similar to those used in clinical practice. Therefore,

concerns existed about the effects of steroid therapy on long-term cognitive

outcome. To examine the potential harmful effect of treatment with adjunctive

dexamethasone on long-term neuropsychological outcome in adults with

bacterial meningitis a follow-up study of the European Dexamethasone Study

was conducted.52 In 87 of 99 eligible patients, 46 (53%) of whom were treated

with dexamethasone and 41 (47%) of whom received placebo, no significant

differences in outcome were found between patients in the dexamethasone

and placebo groups (median time between meningitis and testing was 99

months). Therefore, treatment with adjunctive dexamethasone does not

worsen long-term cognitive outcome in adults after bacterial meningitis.

By reducing permeability of the BBB, steroids can impede penetration of

antibiotics into the CSF, as was shown for vancomycin in animal studies,53 which

can lead to treatment failures, especially in patients with meningitis due to drug-

resistant pneumococci in whom antibiotic regimens often include vancomycin.

However, an observational study, which included 14 adult patients admitted

to the intensive care unit because of suspected pneumococcal meningitis,

appropriate concentrations of vancomycin in CSF were obtained even when

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concomitant steroids were used.54 The dose of vancomycin used in this study

was 60 mg/kg/day. Although these results suggest that dexamethasone can

be used without fear of impeding vancomycin penetration into the CSF of

patients with pneumococcal meningitis (provided that vancomycin dosage is

adequate), it is recommended that patients with bacterial meningitis due to

non-susceptible strains, treated with adjunctive dexamethasone, are carefully

monitored throughout treatment.

We compared 2 cohorts of adults with culture proven bacterial meningitis:

patients from the Dutch Bacterial Meningitis Cohort Study (1998-2002),

before routine treatment with dexamethasone, with patients enrolled in

the MeninGene study (2006-2009).Chapter 4,5 In adults with meningococcal

meningitis, adjunctive dexamethasone did not influence rates of unfavorable

outcome. However, there was a favorable trend for death and hearing loss in

the meningococcal subgroup in the absence of any excess adverse events. We

therefore concluded that when the patient is identified to have meningococcal

meningitis there is no obvious reason to discontinue dexamethasone.

In pneumococcal meningitis, the outcome of adults with community-acquired

pneumococcal meningitis on a national level has significantly improved over

the last few years. We found a decline in unfavorable outcome from 50 to

39%. We used a historical cohort design to evaluate the treatment effect in

pneumococcal en meningococcal meningitis. Unknown differences between

treatment groups may have existed. However, we have corrected for known

prognostic factors and the treatment effect was consistent with the results of

the European Dexamethasone Study.44

Other adjunctive therapies

Glycerol is a hyperosmolar agent that has been used in several neurological and

neurosurgical disorders to decrease intracranial pressure. Although glycerol

has no beneficial effect in experimental meningitis models, a small randomised

clinical trial in Finland suggested that this drug might protect against sequelae

in children with bacterial meningitis.55, 56 A large study in children with bacterial

meningitis in several South American countries showed a significant decrease

in sequelae, but there were several questions about the methodology of the

trial.57 A recent trial in Malawi in adults however showed that adjuvant glycerol

was harmful and increased mortality.58 Therefore, there appears to no role for

treatment with adjuvant glycerol in bacterial meningitis in adults. For children

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the evidence is currently insufficient to justify routine glycerol treatment.

The management of adults with bacterial meningitis can be complex and

common complications are meningoencephalitis, systemic compromise, stroke

and raised intracranial pressure (Figure 2). Various adjunctive therapies have been

described to improve outcome in such patients, including anti-inflammatory

agents, anticoagulant therapies, and strategies to reduce intracranial pressure

(ICP).26 Few randomized clinical studies are available for other adjunctive

therapies than corticosteroids in adults with bacterial meningitis.

Recently a randomized controlled trial was published on adjuvant high dose

paracetamol in children with bacterial meningitis in Luanda. The trial was

performed in a 2x2 design in which simultaneously slow infusion of antibiotics

was compared to bolus injection of antibiotics. No benefit on the primary

endpoints were observed in any of the treatment groups.59

A Dutch cohort study evaluated the effects of complications on mortality in

patients with pneumococcal meningitis and compared these findings among

different age-groups. In older patients (≥60 years), death was usually a result

of systemic complications, whereas death in younger patients (<60 years) was

predominantly due to neurological complications such as brain herniation.29

This observation may be explained by age-related cerebral atrophy, which

allows elderly patients to tolerate brain swelling. These findings suggest that

supportive treatments that aim to reduce ICP could be most beneficial in

younger adults with pneumococcal meningitis. Methods available to reduce

intracranial pressure range from simple (e.g., elevation of the head of the bed

to 30°) to aggressive strategies (e.g., “Lund concept”).26, 60 However, there is no

evidence that ICP monitoring and treatment of increased ICP is beneficial in

patients with bacterial meningitis.

The rationale of hyperventilation in patients with bacterial meningitis is the

relation between cerebral arteriolar dilation, increased cerebral blood flow

(CBF) and a subsequent rise in ICP. Hyperventilation-induced hypocapnia

causes (cerebral) vasoconstriction and a reduction in CBF, resulting in lowering

of ICP.26 This approach has been used in patients with traumatic brain injury

as well; however, the enthusiasm for hyperventilation was greatly tempered

after a study on prophylactic hyperventilation in patients with severe brain

injury showed a worse outcome.26 In bacterial meningitis, patients are often

hypocapneic at the time of admission suggesting that there is spontaneous

hyperventilation.

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In patients with traumatic brain injury, studies have shown that mannitol

decreases blood viscosity and reduces the diameter of pial arterioles in a

manner similar to the vascoconstriction produced by hyperventilation.26

Although osmotic tissue dehydration may still play some role, mannitol works

primarily through its immediate rheological effect, by diluting the blood

and increasing the deformability of erythrocytes, thereby decreasing blood

viscosity ad increased CBF. This sudden increase in CBF causes autoregulatory

vasoconstriction of cerebral arterioles, decreasing the intracerebral blood

volume and lowering ICP. In bacterial meningitis, because BBB permeability has

been increased, the effect of mannitol is uncertain. There is little information

from clinical and experimental studies concerning the use of mannitol in

bacterial meningitis. A single dose of mannitol reduced ICP for approximately

3 hours in a meningitis model.(Lorenz CCM 1996) Continuous intravenous

infusion of mannitol attenuated the increases of regional CBF, brain water

content and ICP in a pneumococcal meningitis model.

Seizures occur frequently in patients with bacterial meningitis.61 These patients

tend to be older, are more likely to have focal abnormalities on brain CT and to

have S. pneumoniae as the causative micro-organism, and they have a higher

mortality. The high mortality warrants a low threshold for starting antiepileptic

therapy in those with clinical suspicion of seizures.

Recurrent bacterial meningitis

Recurrent bacterial meningitis occurs in 5% of community-acquired bacterial

meningitis cases, and most patients have a predisposing condition, particularly

head injury and CSF leak, only occasionally impairment of humoral immunity.62,

63 In patients with no apparent cause of recurrent meningitis or known history

of head trauma, the high prevalence of remote head injury and CSF leakage

justifies an active search for anatomical defects and CSF leakage. Detection of

β-2 transferrine in nasal discharge is a sensitive and specific method to confirm

a CSF leak and thin-slice CT of the skull base is best to detect small bone defects.

It should be kept in mind though that the detection of a small bone defect does

not prove CSF leakage. 3D-CISS MRI images may show CSF flow into the nasal

cavity or paranasal sinuses and may provide additional evidence for CSF leakage.

Surgical repair has a high success rate with low mortality and morbidity.

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Outcome

Community-acquired bacterial meningitis in adults is a severe disease with

high fatality and morbidity rates. Meningitis caused by S. pneumoniae has the

highest case fatality rate, reported from 19 to 37%.1, 2, 8 Whereas neurological

complications are the leading cause of death in younger patients, elderly

patients die predominantly from systemic complications. Of those who

survive, up to 50% develop long-term neurologic sequelae, including

cognitive impairment.64 Hearing loss commonly complicates pneumococcal

meningitis and is present in ~25% of patients. Serotype 23F is associated with a

significant lower risk of hearing loss, as compared with the serotype 3. Otitis on

presentation is a risk factor for developing hearing loss and otolaryngological

evaluation in these patients should be performed on admission. chapter 6

For meningococcal meningitis mortality and morbidity rates are lower, with

rates up to 5 and 7 percent, respectively. The strongest risk factors for an

unfavorable outcome in patients with bacterial meningitis are those indicative

of systemic compromise, impaired consciousness, low cerebrospinal fluid white-

cell count, and infection with S. pneumoniae.30 Recently, we have constructed

and validated a simple model for predicting outcome, using six variables that

are routinely available within one hour of admission.65 This helps to identify

high-risk individuals and provides important information for patients and their

relatives (Figure 3).

Recommendations for future research

Vaccination strategies have greatly reduced the incidence of meningococcal

disease, and the implementation of the PCV7 pneumococcal vaccine has

reduced incidence of pneumococcal meningitis in children and is beginning

to show effect of herd immunity, reducing invasive pneumococcal disease

in adults. The development of additional meningococcal vaccines, such as

meningococcal group B vaccine, will further reduce the burden of disease in

developed countries. However, acces to already existing vaccines in low-income

countries is currently the single most important factor for the reduction of the

global burden of disease in bacterial meningitis.

The importance of national and international scientific collaboration, as

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illustrated in chapters 3 and 7, is essential to the advance of clinical medicine

through translational research. Translational medicine is the integration of

basic and clinical sciences to improve diagnosis and treatment in patients.

Sharing data internationally will contribute to developing new treatments

for bacterial meningitis. As bacterial meningitis continues to cause severe

disease, further research into elucidating the mechanisms of damage caused

by inflammatory response will reveal new strategies for reducing morbidity

and mortality in bacterial meningitis. The implementation of dexamethasone

in high-income countries has been an important step in this process. Further

research into clinical applicabililty of C5-specific monoclonal antibodies is

warranted. Determining of genetic polymorphisms in individual patients may

influence vaccination and treatment strategies in the future.

Figure 3. Prediction rule for risk for unfavorable outcome in adults with bacterial meningitis

% unfavorableoutcome:

TOTAL points:

age: 20 30 40 50 60 70 80

points: 0 2 4 6 8 10 12

points: 0 1 2 4 5 6 8 9 10 12 13 14 16

Glasgow Coma Scale: 15 14 13 12 11 10 9 8 7 6 5 4 3

points: 0 10

tachycardia: No Yes

points: 0 9

cranial nerve palsy: No Yes

points: 0 13

CSF leukocyte count: High Low

points: 0 1 2 12

CSF Gram’s stain: G- No Other G+

0 5 10 15 20 25 30 35 40 45 50 55 60 65

3.2 5.1 8.2 13 20 29 40 52 64 75 83 89 93 96

Tachycardia was defined as a heart rate greater than 120 beats/min. low cerebrospinal fluid (CSF) leukocyte count was defined as <1,000 cells/mm3. Result of CSF Gram’s stain: G– = gram-negative cocci; No = no bacteria; Other = other bacterial species; G+ = grampositive cocci. Instruction: Locate the age of the patient on the top axis and determine how many points the patient receives. Repeat this for the remaining five axes. Sum the points for all six predictors and locate the total sum on the total point axis. Draw a line straight down to the axis labeled “% unfavourable outcome” to find the estimated probability of an unfavourable outcome for this patient.

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46. van de Beek D, de Gans J, McIntyre P, Prasad K. Steroids in adults with acute bacterial meningitis: a systematic review. Lancet Infect Dis 2004;4(3):139-143.

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51. Cronin L, Cook DJ, Carlet J et al. Corticosteroid treatment for sepsis: a critical appraisal and meta-analysis of the literature. Crit Care Med 1995;23(8):1430-1439.

52. Weisfelt M, Hoogman M, van de Beek D, de Gans J, Dreschler WA, Schmand BA. Dexamethasone and long-term outcome in adults with bacterial meningitis. Ann Neurol 2006;60(4):456-468.

53. Paris MM, Hickey SM, Uscher MI, Shelton S, Olsen KD, McCracken GH, Jr. Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrob Agents Chemother 1994;38(6):1320-1324.

54. Ricard JD, Wolff M, Lacherade JC et al. Levels of vancomycin in cerebrospinal fluid of adult patients receiving adjunctive corticosteroids to treat pneumococcal meningitis: a prospective multicenter observational study. Clin Infect Dis 2007;44(2):250-255.

55. Kilpi T, Peltola H, Jauhiainen T, Kallio MJ. Oral glycerol and intravenous dexamethasone in preventing neurologic and audiologic sequelae of childhood bacterial meningitis. The Finnish Study Group. Pediatr Infect Dis J 1995;14(4):270-278.

56. Singhi S, Jarvinen A, Peltola H. Increase in serum osmolality is possible mechanism for the beneficial effects of glycerol in childhood bacterial meningitis. Pediatr Infect Dis J 2008;27(10):892-896.

57. Peltola H, Roine I, Fernandez J et al. Adjuvant glycerol and/or dexamethasone to improve the outcomes of childhood bacterial meningitis: a prospective, randomized, double-blind, placebo-controlled trial. Clin Infect Dis 2007;45(10):1277-1286.

58. Ajdukiewicz KM, Cartwright K, Scarborough M et al. A double blind, randomised controlled trial of glycerol adjuvant therapy in adult bacterial meningitis in a high HIV seroprevalence setting in Malawi. Lancet Infect Dis 2011;11(4):293-300.

59. Pelkonen T, Roine I, Cruzeiro ML, Pitkaranta A, Kataja M, Peltola H. Slow initial beta-lactam infusion and oral paracetamol to treat childhood bacterial meningitis: a randomised, controlled trial. Lancet Infect Dis 2011;11(8):613-21.

60. Grande PO. The “Lund Concept” for the treatment of severe head trauma--physiological principles and clinical application. Intensive Care Med 2006;32(10):1475-1484.

61. Zoons E, Weisfelt M, de Gans J et al. Seizures in adults with bacterial meningitis. Neurology 2008;70(22 Pt 2):2109-2115.

62. Adriani KS, van de Beek D, Brouwer MC, Spanjaard L, de Gans J. Community-acquired recurrent bacterial meningitis in adults. Clin Infect Dis 2007;45(5):e46-e51.

63. Tebruegge M, Curtis N. Epidemiology, etiology, pathogenesis, and diagnosis of recurrent bacterial meningitis. Clin Microbiol Rev 2008;21(3):519-537.

64. van de Beek D, Schmand B, de Gans J et al. Cognitive impairment in adults with good recovery after bacterial meningitis. J Infect Dis 2002;186(7):1047-1052.

65. Weisfelt M, van de Beek D, Spanjaard L, Reitsma JB, de Gans J. A risk score for unfavorable outcome in adults with bacterial meningitis. Ann Neurol 2008;63(1):90-7.

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Summary

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176

Bacterial meningitis is a life-threatening infectious disease. Two common

inhabitants of the human nasopharynx, Streptococcus pneumoniae and Neisseria

meningitidis, are the most prevalent causes of bacterial meningitis. When

bacteria invade the membranes lining the brain, meningitis occurs. Effective

antibiotic treatment and adequate critical care have improved the prognosis

greatly, but mortality remains substantial and in survivors neurologic sequelae

such as hearing loss are common.

In chapter 2 of this thesis we describe 258 patients with meningococcal

meningitis who were included in a nationwide prospective cohort study from

1998-2002. On presentation, the prevalence of the classical triad of fever,

neck stiffness, and change in mental status was low (27%). Rash, a common

presenting symptom of meningococcal disease, was found in 64% of patients.

When septic shock occurs, hypotension and tachycardia are common and these

were found 31% of patients. The clinical importance of identifying a rash in

meningococcal disease was underlined by the presence of rash in all 5 patients

with normal initial cerebrospinal fluid examination. Bacterial genotyping

was performed through MLST analysis and disease caused by meningococci

belonging to clonal complex 11 was associated with sepsis and poor outcome.

Sepsis was the leading cause of mortality.

In chapter 3, the collaborative effort of our research group with the Netherlands

Vaccine Institute is described. In Gram-negative bacteria such as N. meningitidis,

lipopolysaccharide is a major component of the outer membrane, and lipid A

is an important part of that lipopolysaccharide. The human immune system

recognizes lipid A through binding with Toll-like receptor 4 (TLR4), which induces

activation of the innate immune system and induction of cytokine production.

A meningococcal strain with reduced TLR4 activation was investigated

and found to have a changed lipid A structure, caused by mutations in the

meningococcal lpxL1 gene. We found these mutations in 7% of meningococci

from the patients in our prospective cohort study and investigated if clinical

characteristics in these patients were different. In these patients, we found

that rash and reduced platelet counts were less common, consistent with less

systemic inflammation and reduced activation of the coagulant system. These

results provide the first example of a specific mutation in N. meningitidis that

can be correlated with the clinical course of meningococcal meningitis.

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Sum

ma

ry

In 2002, a European randomized clinical trial showed beneficial effect of

adjunctive dexamethasone in adults with bacterial meningitis. The effect

was most pronounced in pneumococcal meningitis and mortality in those

patients was reduced by 10%. While these results were not reproduced

in clinical trials from other parts of the world, an individual patient data-

analysis and subsequent Cochrane review supported the continued use of

dexamethasone in children and adults in high-income countries. However,

the use of adjunctive dexamethasone in meningococcal meningitis remained

controversial, and some international guidelines recommended discontinuing

dexamethasone in suspected meningococcal meningitis. In clinical practice,

Gram staining of cerebrospinal fluid is frequently negative and the results of

cultures requires several days. Furthermore, lumbar puncture may be contra-

indicated, prohibiting CSF analysis. Therefore, the cause of bacterial meningitis

may remain unknown in the first days of treatment, when dexamethasone

is administered. In chapters 4 and 5, we used a historical cohort design to

compare the prognosis of patients with meningococcal and pneumococcal

meningitis in 2 cohorts, one from 1998-2002, before the implementation of

adjunctive dexamethasone, and one from 2006-2009 after the introduction

of adjunctive dexamethasone. In chapter 4, we showed that the prognosis of

meningococcal meningitis has not changed since the introduction of adjunctive

dexamethasone treatment. We found no reason to discontinue the use of

adjunctive dexamethasone in these patients. In chapter 5, we studied the effect

of adjunctive dexamethasone in pneumococcal meningitis. The prognosis of

patients with pneumococcal meningitis has improved substantially since the

introduction of bacterial meningitis. The absolute reduction in both mortality

(from 30% to 20%) and unfavorable outcome (from 50% to 39%) could not be

attributed to other factors, such as changes in epidemiology or disease severity.

These studies support the continued use of adjunctive dexamethasone in

adults with bacterial meningitis in the Netherlands.

In patients that survive bacterial meningitis, neurologic sequelae are common

and include cognitive impairment and hearing loss. In chapter 6 we studied

the incidence and severity of hearing loss in patients surviving pneumococcal

meningitis. Otitis was a common presenting feature (36% of patients) and

predictive of hearing loss at discharge. Hearing loss was most common in

patients infected with pneumococcal serotype 3.

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In patients with bacterial meningitis, the improvement in outcome with

adjunctive dexamethasone is based on reduction of the inflammatory response.

The complement system is an important part of the innate immune system and

reducing the proinflammatory effect of complement activation may further

improve prognosis. In chapter 7, we found that common genetic variants in the

complement system influence outcome. Patient with pneumococcal meningitis

with the GG genotype in a gene coding or complement factor 5 (rs17611) were

at higher risk for unfavorable outcome. In a mouse model, treatment with

C5a antibodies improved outcome in pneumococcal meningitis and further

research of this new adjuvant treatment is warranted.

In chapter 8 we discuss the epidemiology, pathophysiology and treatment of

bacterial meningitis and provide context for the findings of our research and

recommendations for future research. Improving the prognosis of patients with

bacterial meningitis remains an important challenge. We have shown that both

bacterial genotype and human genetic variants influence disease course and

outcome in this life-threatening disease. While treatment with dexamethasone

has reduced unfavorable outcome in the Netherlands substantially, further

research is warranted to continue improving the prognosis of patients with

bacterial meningitis.

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Samenvatting

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Bacteriële meningitis (hersenvliesontsteking, soms ook nekkramp genoemd)

is een levensbedreigende infectieziekte. De belangrijkste veroorzakers

zijn Streptococcus pneumoniae (pneumokokken) en Neisseria meningitidis

(meningokokken), bacteriën die veel gezonde mensen bij zich dragen in de

mond- en keelholte. Wanneer bacteriën de hersenvliezen bereiken ontstaat

hersenvliesontsteking. De prognose van deze ziekte is sterk verbeterd sinds

er effectieve antibiotica zijn en er goede ondersteunende zorg is, zoals op een

intensive care. Desondanks overlijden nog steeds 20% van deze patiënten

en houden patiënten die de ziekte overleven vaak restverschijnselen, zoals

gehoorsverlies.

In hoofdstuk 2 van dit proefschrift beschrijven we een studie bij 258 patiënten

met meningokokkenmeningitis uit een landelijk, prospectief onderzoek van

1998-2002. De klassieke trias van koorts, nekstijfheid en verlaagd bewustzijn

was bij slechts 27% van de patiënten aanwezig. Purpura of petechiën (rode, niet

wegdrukbare plekken in de huid) waren aanwezig bij 64% van de patiënten.

Tekenen van sepsis (bloedvergiftiging door bacteriën in de bloedbaan)

werden gevonden bij 31% van de patiënten. Het is belangrijk om de typische

huidafwijkingen te herkennen, ook omdat bij de 5 patiënten in deze studie die

geen afwijkingen in het hersenvocht hadden, de ziekte bij alle 5 te herkennen

was aan de huiduitslag. Genotypering van de bacteriën middels MLST analyse

werd verricht en meningokokken van een bepaalde “familie” (clonal complex

11) veroorzaakten vaker sepsis met tevens een slechtere prognose.

In hoofdstuk 3 beschrijven we de resultaten van een gezamenlijk project

met het Nederlands Vaccin Instituut. Bij gram-negatieve bacteriën zoals

meningokokken is het lipopolysaccharide een belangrijk onderdeel van

de celmembraan, en het lipide A is hier onderdeel van. Het menselijke

immuunsysteem herkent het lipide A doordat het bindt met Toll-like receptor 4

(TLR4), waarna er activatie van het aangeboren immuunsysteem met cytokine

productie plaatsvindt. Een meningokok met een verminderd vermogen tot

TLR4-activatie is nader onderzocht en bleek een afwijkende structuur van

lipide A te hebben, veroorzaakt door een mutatie in het lpxL1-gen van de

meningokok. Vervolgens onderzochten we de meningokokken afkomstig van

meningitispatiënten en bleek er in 7% van de bacteriën een mutatie in het lpxL1-

gen aanwezig te zijn. Verdere analyse toonde aan dat deze patiënten minder

vaak huiduitslag en vermindering van bloedplaatjes in het bloed hadden,

passend bij een minder hevige immuunrespons en verminderde activatie

24407 Heckenberg.indd 182 25-02-13 11:12

183

Sam

enva

ttin

g

Samenvatting

van het stollingssysteem. We hebben zo voor het eerst aangetoond dat een

specifieke mutatie in meningokokken het beloop van de ziekte beïnvloedt.

In 2002 werden de resultaten van een Europese studie gepubliceerd, waaruit

bleek dat behandeling met dexamethason de prognose van patiënten met

bacteriële meningitis verbetert. Bij patiënten met pneumokokkenmeningitis

daalde de sterfte van 30% naar 20%. Hierna werden vergelijkbare onderzoeken

in andere delen van de wereld gedaan, maar de resultaten weren niet

gereproduceerd. De resultaten van verschillende onderzoeken zijn gebundeld

in meta-analyses en deze ondersteunden het gebruik van dexamethason in

Nederland. Bij patiënten met meningokokkenmeningitis bleef het gebruik

van dexamethason echter omstreden en sommige richtlijnen adviseerden

deze patiënten niet met dexamethason te behandelen. In de praktijk komt

het regelmatig voor dat bij de start van de behandeling de verwekker nog

niet bekend is. De gram-kleuring van het hersenvocht levert frequent geen

verwekker op en de resultaten van kweken laten enkele dagen op zich wachten.

Ook kunnen er redenen zijn om geen lumbaalpunctie te verrichten waardoor

de verwekker moeilijker te identificeren is. In hoofdstuk 4 en 5 hebben we

gegevens van 2 cohorten met elkaar vergeleken, een van 1998-2002, voordat

de behandeling met dexamethason werd ingevoerd, en een van 2006-2009, na

de invoering van dexamethason. In hoofdstuk 4 tonen we aan dat de prognose

van meningokokkenmeningitis niet veranderd is sinds de invoering van

dexamethason en we geen reden hebben gevonden om de behandeling met

dexamethason bij deze patiënten te ontraden. In hoofdstuk 5 beschrijven we

de verandering van de prognose bij patiënten met pneumokokkenmeningitis

sinds de invoering van de behandeling van dexamethason. De prognose

van patiënten met pneumokokkenmeningitis is duidelijk verbeterd sinds de

invoering van dexamethason. De afname van sterfte van 30% naar 20% en de

vermindering van patiënten met ongunstige uitkomst van 50% naar 39% kon

niet toegeschreven worden aan andere oorzaken, zoals veranderingen in de

ernst van de ziekte of andere verschuivingen. Deze resultaten ondersteunen

het gebruik van dexamethason bij patiënten met zowel meningokokken- als

pneumokokkenmeningitis.

Bij patiënten die bacteriële meningitis overleven blijken vaak restverschijnselen

aanwezig te zijn, zoals cognitieve stoornissen en gehoorsverlies. In hoofdstuk

6 beschrijven we de ernst en frequentie van gehoorsverlies bij patiënten

die een pneumokokkenmeningitis overleefden. Oorontstekingen zijn vaak

24407 Heckenberg.indd 183 25-02-13 11:12

184

al bij opname in het ziekenhuis aanwezig en vormen een risicofactor voor

gehoorsverlies. Pneumokokken van serotype 3 veroorzaakten het vaakst

gehoorsverlies.

De verbeterde uitkomst bij bacteriële meningitis door behandeling

met dexamethason wordt veroorzaakt door het verminderen van de

ontstekingsreactie. Het complement systeem is een belangrijk onderdeel

van het immuunsysteem en vermindering van de ontstekingsreactie door

remming van het complementsysteem kan mogelijk de prognose van

bacteriële meningitis verbeteren. In hoofdstuk 7 tonen we aan dat kleine,

veelvoorkomende varianten van de genen betrokken bij het complement

systeem de prognose beïnvloeden. Patiënten met het GG fenotype in

het rs17611 gen, dat codeert voor complement factor C5, hebben bij

pneumokokkenmeningitis een slechtere prognose. Uit verder onderzoek in

een model van bacteriële meningitis in muizen bleek dat behandeling met

een antilichaam tegen C5a de prognose verbetert. Verder onderzoek naar deze

behandeling zal duidelijk moeten maken of deze toepasbaar is.

In hoofdstuk 8 beschrijven we de epidemiologie, pathofysiologie en behandeling

van bacteriële meningitis en plaatsen we onze onderzoeksresultaten in een

bredere context. De verdere verbetering van de prognose van patiënten met

bacteriële meningitis blijft een belangrijke uitdaging. We hebben aangetoond

dat de erfelijke eigenschappen van zowel de bacteriën als de patiënten een rol

spelen bij het beloop van de ziekte en de prognose van deze patiënten. Hoewel

de behandeling met dexamethason in Nederland de uitkomst van patiënten

heeft verbeterd, is verder onderzoek naar nieuwe behandelingen nodig om de

prognose van patiënten met bacteriële meningitis verder te verbeteren.

24407 Heckenberg.indd 184 25-02-13 11:12

24407 Heckenberg.indd 185 25-02-13 11:12

24407 Heckenberg.indd 186 25-02-13 11:12

Dankwoord

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188

Graag wil ik de volgende mensen in het bijzonder danken voor hun bijdrage

aan dit proefschrift.

Diederik, ik ben zeer vereerd je eerste officiële promovendus te zijn in wat

ongetwijfeld een lange reeks zal worden. Dank voor je begeleiding, je altijd

positieve inslag bij tegenslagen, je humor en natuurlijk de teambuilding tijdens

de congressen.

Matthijs, ik heb met veel plezier met je samengewerkt. Je weet elk project

tot een succes te maken en naast het werk hebben we veel gelachen op H2,

maar ook op de dansvloer in Chicago en de pistes in Frankrijk. Ik ben blij dat

je me bij wilt staan als paranimf en ben ervan overtuigd dat je een zonnige

wetenschappelijke toekomst tegemoet gaat in het AMC.

Jan, na een inspirerend gesprek met jou was mijn enthousiasme voor

het meningitisonderzoek gewekt. Dank voor het warme welkom in de

meningitisclub.

Rien, je hebt het meningitis onderzoek altijd van harte ondersteund en ik heb

veel van je geleerd van je tijdens de opleiding. Dank daarvoor.

De overige promovendi van de immer uitdijende meningitisclub: Barry,

Madelijn, Jurgen, Ewout, Kirsten, dank voor de teamgeest, de gezelligheid op

H2 en tijdens de congressen. Ik hoop jullie binnenkort te zien promoveren.

Barry, dank voor de bird’s-eye view en dat je me wilt bijstaan als paranimf.

De studenten die hebben geholpen met het onderzoek: Kinki Jim, Soemirien

Kasanmoentalib, Daan Fritz en Floortje Ruiter.

De medewerkers van het Nederlands Referentielaboratorium voor Bacteriële

Meningitis: Agaath Arens-van ‘t Klooster, Wendy Keijzers, Virma Godfried,

Yvonne Pannekoek en Arie van der Ende.

De medewerkers van het laboratorium voor genoomanalyse: Mia van Meegen,

Marit de Wissel, Jeroen Vreijling, Laurens Bordewijk, Suzanne Kenter, Ruud

Wolterman en Frank Baas.

24407 Heckenberg.indd 188 26-02-13 09:17

189

Da

nkw

oo

rD

DankwoorD

Alle neurologen, arts-assistenten, IC-artsen, infectiologen en microbiologen

die het onderzoek mogelijk maken en alle bacteriële meningitis patiënten,

partners en familieleden voor de bereidheid aan ons onderzoek mee te werken.

Alle mede-auteurs, dank voor de vruchtbare samenwerking en de kritische

kanttekeningen.

Mijn collega’s in het Kennemer Gasthuis. Ik heb het met jullie getroffen en als

jonge maatschap ligt de toekomst aan onze voeten!

Niels, Klarke, Mark, Sanne, Ard, Diedeke, Miranda, Tijmen, Ruben, Doris, Esther,

Orian, Baars, Heidi en Luuk. Dank voor jullie vriendschap, humor, steun, leuke

avonden en vakanties samen.

My father Dick, thank you for your support.

Mijn broer Jason, Tineke, zwager Wim en Désirée, dank voor jullie interesse en

afleiding tijdens dit lange project.

Mijn schoonouders Jaap en Gré, betere schoonouders kan ik mij niet wensen!

Dank voor jullie ondersteuning, zowel in emotionele als praktische zin.

Mijn moeder Mariëtte. Lieve moeders, dank voor je nimmer aflatende steun in

alles wat ik wil bereiken (behalve dan het halen van mijn vliegbrevet…).

Als laatste mijn onmisbare thuisbasis. Lieve Thijs en Nadine, elke dag delen

we plezier en geluk, en als vader barst ik bijna uit elkaar van trots. Tenslotte

mijn lief Marjolein, in de soms hectische afgelopen jaren heb je me altijd

onvoorwaardelijk gesteund als ik weer eens aan dit boekje moest werken.

Zonder jouw liefde en steun was het me nooit gelukt. Laten we samen genieten

van “het leven na de promotie”… ik heb er zin in!

24407 Heckenberg.indd 189 25-02-13 11:12

24407 Heckenberg.indd 190 25-02-13 11:12

Curriculum vitae

24407 Heckenberg.indd 191 25-02-13 11:12

192

Sebastiaan Heckenberg werd op zondag 5 juni 1977 geboren te Amsterdam,

als 2e zoon van een Nederlandse moeder en een Amerikaanse vader. Na de

lagere school te Vinkeveen en het VWO op het Alkwin Kollege te Uithoorn

begon hij in 1995 de studie geneeskunde aan de VU te Amsterdam. Tijdens

zijn studie werkte hij als student-assistent op de afdeling fysiologie en was hij

in 1999 bestuurslid van de Medische Faculteitsvereniging. Hij deed met Ruben

Schouten een wetenschappelijke stage in de streek rond Ellisras, Zuid-Afrika.

Na het behalen van het doctoraal startte in 2000 de co-schappen en liep hij

in 2002 met Marjolein een co-schap op de spoedeisende hulp van het West

Middlesex University Hospital te Londen. Vervolgens begon hij in 2003 als

AGNIO op de adeling neurologie van het Onze Lieve Vrouwe Gasthuis waarna

de opleiding neurologie in 2004 in het AMC begon. Vanaf 2005 combineerde

hij de opleiding met het meningitis onderzoek. Sinds april 2011 is hij werkzaam

als neuroloog in het Kennemer Gasthuis te Haarlem. Hij heeft met zijn vriendin

Marjolein twee kinderen, Thijs en Nadine.

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24407 Heckenberg.indd 194 25-02-13 11:12

List of publications

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196

Heckenberg SG, Brouwer MC, van de Beek D. Bacterial meningitis: epidemiology,

pathophysiology and treatment. Handbook of Clinical Neurology 2013 (in

press).

Heckenberg SG, Brouwer MC, van der Ende A, van de Beek D. Adjunctive

dexamethasone in adults with meningococcal meningitis. Neurology,

2012;79(15):1563-9.

Heckenberg SG, Brouwer MC, van der Ende A, Hensen EF, van de Beek D. Hearing

loss in adults surviving pneumococcal meningitis is associated with otitis and

pneumococcal serotype. Clinical Microbiology and Infection, 2012;18(9):849-

55.

Brouwer MC, Heckenberg SG, van Well GTJ, Brouwer A, Delwel EJ, Spanjaard

L, van de Beek D, Prins JM. SWAB Guidelines on Antibacterial Therapy of

Patients with Bacterial Central Nervous System Infections. Stichting Werkgroep

Antibioticabeleid 2012.

Woehrl B, Brouwer MC, Murr C, Heckenberg SG, Baas F, Pfister HW, Zwinderman

AH, Morgan BP, Barnum SR, van der Ende A, Koedel U, van de Beek D.

Complement component 5 contributes to poor disease outcome in humans

and mice with pneumococcal meningitis. Journal of Clinicical Investigation,

2011;121(10):3943-53.

Brouwer MC, Heckenberg SG, de Gans J, Spanjaard L, Reitsma JB, van de Beek

D. Nationwide implementation of adjunctive dexamethasone therapy for

pneumococcal meningitis. Neurology, 2010;75(17):1533-9.

Fransen F, Heckenberg SG, Hamstra HJ, Feller M, Boog CJ, van Putten JP,

van de Beek D, van der Ende A, van der Ley P. Naturally occurring lipid A

mutants in neisseria meningitidis from patients with invasive meningococcal

disease are associated with reduced coagulopathy. PLoS Pathogens. 2009

Apr;5(4):e1000396.

Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van

de Beek D. Host genetic susceptibility to pneumococcal and meningococcal

24407 Heckenberg.indd 196 25-02-13 11:12

197

Publ

icat

ion

S

LiSt of pubLicationS

disease: a systematic review and meta-analysis. Lancet Infectious Diseases,

2009;9(1):31-44.

Brouwer MC, de Gans J, Heckenberg SG, Kuiper H, van Lieshout HB, van de Beek

D. Vertebral osteomyelitis complicating pneumococcal meningitis. Neurology,

2008;71(8):612-3.

Heckenberg SG, de Gans J, Brouwer MC, Weisfelt M, Piet JR, Spanjaard L, van

der Ende A, van de Beek D. Clinical features, outcome, and meningococcal

genotype in 258 adults with meningococcal meningitis: a prospective cohort

study. Medicine (Baltimore), 2008;87(4):185-92.

Brouwer MC, van de Beek D, Heckenberg SG, Spanjaard L, de Gans J. Community-

acquired Haemophilus influenzae meningitis in adults. Clinical Microbiology

and Infection, 2007;13(4):439-42.

Brouwer MC, van de Beek D, Heckenberg SG, Spanjaard L, de Gans J.

Hyponatraemia in adults with community-acquired bacterial meningitis. QJM,

2007;100(1):37-40.

Brouwer MC, van de Beek D, Heckenberg SG, Spanjaard L, de Gans J. Community-

acquired Listeria monocytogenes meningitis in adults. Clinical Infectious

Diseases, 2006;43(10):1233-8.

24407 Heckenberg.indd 197 25-02-13 11:12

Bacterial m

enin

gitis in

adu

lts Sebastiaan

G.B

. Hecken

berg


Host and pathogen factors, treatment and outcome


24407 Heckenberg_omslag 24-02-13 15:36 Pagina 1

Stellingen behorende bij het proefschrift

Bacterial meningitis in adults:

host and pathogen factors, treatment and outcome

Meningococcal genotype influences disease course and outcome in meningococcal meningitis. This thesis.

The introduction of adjunctive treatment with dexamethasone has improved the prognosis in patients with pneumococcal meningitis in the Netherlands. This thesis.

In patients with meningococcal meningitis, treatment with adjunctive dexamethasone can be safely administered. This thesis.

Genome-wide association studies are useful in identifying new pathways for adjunctive treatment in bacterial meningitis. This thesis.

Science benefits more from cooperation than from competition.

Cynicism is a sorry kind of wisdom. Barack Obama.

If we amplify everything, we hear nothing. Jon Stewart.

The problem with the future is that it keeps turning into the present. Bill Watterson.

Wie denkt dat kennis duur is, weet niet wat domheid kost. Alexander Rinnooy Kan.

Open access heeft de toekomst.

24407 Heckenberg stellingen.indd 1 08-03-13 15:35

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