Skip Navigation


JAC Advance Access originally published online on October 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1291-1294; doi:10.1093/jac/dkl401
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/6/1291    most recent
dkl401v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Brandt, C. T.
Right arrow Articles by Østergaard, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandt, C. T.
Right arrow Articles by Østergaard, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Evaluation of anti-pneumococcal capsular antibodies as adjunctive therapy in experimental pneumococcal meningitis

Christian T. Brandt1,*, Niels Frimodt-Møller1, Jens D. Lundgren2, Michael Pedersen3, Ian C. Skovsted1, Ian J. Rowland4 and Christian Østergaard1,5

1 National Center for Antimicrobials and Infection Control, Statens Serum Institut Artillerivej 5, 2300 Copenhagen S, Denmark 2 CHIP, Copenhagen University Hospital Hvidovre Kettegaard Allé 30, 2650 Hvidovre, Denmark 3 Department of Infectious Diseases, Copenhagen University Hospital Rigshospitalet 2100 Copenhagen, Denmark 4 Danish Research Centre for Magnetic Resonance, Copenhagen University Hospital Hvidovre Kettegaard Allé 30, 2650 Hvidovre, Denmark 5 Department of Clinical Microbiology, Copenhagen University Hospital Herlev Herlev Ringvej, 2730 Herlev, Denmark

*Corresponding author. Tel: +45-32-68-81-83/+45-22-91-44-49; Fax: +45-32-68-32-31; E-mail: ctb{at}ssi.dk

Received 30 May 2006; returned 19 July 2006; revised 6 September 2006; accepted 7 September 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objective: Bacteraemia concomitant with meningitis has been shown to greatly affect outcome. Consequently, the efficacy of serotype-specific anti-pneumococcal antiserum (APAS) was investigated in a rat model of pneumococcal meningitis.

Methods: Rats were infected with Streptococcus pneumoniae serotype 3. All rats received ceftriaxone starting 26 h post-infection. APAS was administered either at the time of infection or 26 h post-infection and effects were compared with rats treated with antibiotics only.

Results and conclusion: A significant clinical benefit was found when APAS was given at the time of infection whereas no effect was found when administered 26 h after infection. This work indicates that the clinical value of using APAS in pneumococcal meningitis may be limited.

Keywords: Experimental meningitis , pneumococcal anti-serum , sepsis , outcome


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Pneumococcal meningitis is characterized by extensive meningeal inflammation, brain oedema, elevated intracranial pressure, seizures, infarction and, potentially, herniation of the brain. Consequently, adjunctive therapeutic approaches have primarily targeted the cerebral manifestations of the disease. However, patients with pneumococcal meningitis do also have bacteraemia in 70% of cases as well as systemic complications (e.g. septic shock, multi-organ failure), which is reported to account for up to 50% of the mortality in patients.1–3 Furthermore, a post hoc analysis of the European Dexamethasone in Adulthood Bacterial Meningitis Study4 showed that the beneficial effect of adjunctive therapy with dexamethasone was due to a reduction of systemic rather than neurological complications suggesting that new adjunctive therapies should also be directed against systemic complications to improve disease outcome.

In the pre-antibiotic era, the use of serotype-specific anti-serum therapy was shown to reduce mortality due to pneumococcal pneumonia and bacteraemia.5 More importantly, combined therapy with anti-serum and antibiotics also reduced mortality in experimental pneumococcal bacteraemia,6 making treatment with serotype-specific anti-serum an interesting adjunctive pneumococcal meningitis therapy.

Therefore, the aim of the present study was to investigate (i) the effect of serotype-specific anti-serum on the development of meningitis and (ii) the effects on outcome of adjunctive treatment with serotype-specific anti-serum in combination with antibiotics.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Experimental meningitis

All experimental procedures were approved by the Danish Animal Inspectorate (Dyreforsoegstilsynet). A Streptococcus pneumoniae type 3 strain [68034, Statens Serum Institut (SSI), Copenhagen, Denmark] was suspended in beef broth (SSI) to ~1 x 106 cfu/mL, and meningitis induced as described previously.7 In brief, young adult male Wistar rats (180–200 g) were anaesthetized with Hypnorm®/Dormicum® and infected by intracisternal injection of 30 µL of the bacterial suspension. Saline (3 mL x 3 times daily) was injected subcutaneously during the period of severe illness until cessation of weight loss. Randomization of rats to the experimental treatment groups was blinded to investigators.

Serotype-specific anti-serum

A serotype-specific polyclonal rabbit anti-pneumococcal capsular serotype 3 antiserum (Pneumosera, Statens Serum Institut, Copenhagen, Denmark, Ref. 16746, Lot No: C313C1, product information available at http://www.ssi.dk/sw1366.asp) was purified (11.8 mg pneumococcal serotype 3 antibody/mL) and diluted 1:1 in PBS. Undiluted antiserum titre, estimated by Neufeld capsule reaction, was 32. All rats treated with anti-pneumococcal antiserum (APAS) received a single intravenous injection of 1.4 µL/g bodyweight.

Study design

Development of meningitis.. Nine rats were infected with a bacterial inoculum of 4.8 x 101 S. pneumoniae. Five rats were treated with APAS at the time of infection, 4 rats served as untreated controls. CSF and blood samples were obtained at 48 and 70 h after infection. A low inoculum was used in order to determine whether APAS could prevent the development of meningitis and to assess durability.

Treatment efficacy.. Seventy-three rats were infected with a bacterial inoculum of ~6 x 104 cfu S. pneumoniae. All rats received ceftriaxone (Rocephalin®, F. Hoffmann-la Roche, Basel, Switzerland, 150 mg/kg subcutaneously x 1 daily for 3 days) initiated 26 h after infection in order to obtain mortality rates comparable to those in adult patients.1

The treatment efficacy of adjunctive therapy with APAS was evaluated in two experimental setups. (i) Pretreatment: APAS was administered at the time of infection (n = 11) and 12 rats served as controls. Antibiotic treatment was initiated 26 h after infection. (ii) Late treatment: APAS was administered 10–12 min prior to antibiotic treatment initiated at 26 h after infection (n = 25) and 25 rats served as controls.

CSF and blood samples were obtained 25 h after infection in both the pretreatment and late-treatment study.

Assessment of outcome and disease scores

Clinical and motor performance scores (Table 1) were assessed three times daily during the acute disease phase (22–72 h after infection) and once daily after that. Disease outcome was graded as follows: (i) Terminally ill; (ii) Motor sequelae for animals with complete/partial limb paralysis/spastic paresis; (iii) Circling rats with permanent skewed positioning of the head suggesting labyrinthitis sequelae; (iv) Normal—rats without signs of sequelae.


View this table:
[in this window]
[in a new window]

  		Table 1. Clinical score and motor performance score guidelines

 
CSF and blood analysis

WBC counts were measured on an automatic cell counter (Medonic Vet-Autocounter 620, Boule Medical, Sweden), and bacterial counts were measured by plating 10-fold serial dilutions of CSF and 50 µL of undiluted blood and a 10-fold dilution of blood.

Statistical analysis

Results are presented as medians and quartiles. Log10 transformation of bacterial counts was performed for analysis. Comparisons between groups were performed by Mann–Whitney U-test (continuous data), Two-way ANOVA (continuous data, repeated measures), Fisher's exact test and log-rank test (categorical data). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
Effect of pretreatment with APAS on the development of meningitis

Untreated rats (n = 4) were terminally ill at 58, 59.8, 70 and 72 h after infection whereas APAS pretreatment (n = 5) prevented animals becoming terminally ill until 72 h after infection (Log-rank test, P = 0.003). CSF bacterial count was decreased in APAS pretreated rats compared with controls at both 48 and 70 h after infection [48 h: 3.5 x 105 cfu/mL (7.0 x 104–2.1 x 106) versus 8.0 x 105 cfu/mL (4.6 x 105–3.9 x 106) and 70 h: 1.9 x 106 cfu/mL (1.4 x 106–2.3 x 107) versus 1.1 x 108 cfu/mL (6.3 x 108–1.6 x 108), Two-way ANOVA, 48 and 70 h, P = 0.01]. APAS pretreatment also prevented the development of bacteraemia at 48 h compared with controls [0 x 101 cfu/mL (0 x 101–0 x 101) versus 6.4 x 102 cfu/mL (9.0 x 101–6.6 x 103)] and only one APAS treated rat developed bacteraemia at 70 h after infection [0 x 101 cfu/mL (0 x 101–3.4 x103) versus 4.2 x 102 cfu/mL (2.4 x 102–6.0 x 102), Two-way ANOVA, 48 and 70 h, P = 0.02].

Effects of pretreatment with APAS on bacterial counts, WBC counts and outcome of meningitis

All rats treated with APAS from time of infection (pretreatment, n = 11) survived, whereas 7 of 12 rats treated only with antibiotics became terminally ill (Figure 1a; Log-rank test, P = 0.003).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. (a) Log-rank test for survival was significantly improved in rats immunized with anti-pneumococcal antiserum from the time of infection compared with controls (P = 0.003). (b) Anti-pneumococcal anti-serum administered at the same time as antibiotic treatment did not affect survival compared with controls (P = 0.97). In both (a and b), the vertical solid black arrow indicates time for initiation of antibiotic treatment (26 h after infection).

 
Bacterial counts in CSF and blood obtained 25 h after infection were significantly attenuated by APAS pretreatment [CSF: 1.1 x 105 cfu/mL (7.0 x 104–5.2 x 106) versus 2.1 x 106 cfu/mL (9.2 x 105–1.4 x 107), Mann–Whitney, P = 0.024, and blood: 0 x 101 cfu/mL (0 x 101–3.0 x 101) versus 4.7 x 102 cfu/mL (1.7 x 102–1.2 x 103), P = 0.0003].

WBC counts in CSF and blood obtained 25 h after infection were not significantly affected by APAS pretreatment [CSF: 1500 (750–3300) versus 2500 (850–6150) x 106 cells/L, P = 0.45 and blood: 6.5 (5.7–8.7) versus 8.0 (6.9–10.3) x 109 cells/L, P = 0.26].

Clinical score and motor performance score were significantly decreased in APAS pretreated rats compared with controls from 0 to 168 h after infection (Two-way ANOVA, P < 0.0001 and P < 0.0001, respectively).

Effects of adjunctive treatment with APAS on the outcome of meningitis

Initiation of APAS treatment together with antibiotics at 26 h after infection did not improve survival compared with rats treated with antibiotics only (Figure 1b; 64%, 16/25 versus 68%, 17/25, log-rank test, P = 0.97).

Final outcome in survivors was not improved in APAS late-treated rats compared with controls (3/17 versus 3/16 without sequelae; 2/17 versus 0/16 with motor sequelae; 8/17 versus 12/16 with labyrinthitis sequelae, Fisher's exact test P > 0.05).

Clinical score and motor performance score (Figure 2a and b) were comparable between groups prior to treatment randomization at 22 h after infection (Mann–Whitney, P = 0.98 and P = 0.99). No differences in clinical appearance and motor performance were observed between the study groups after initiation of the experimental treatments (30–168 h after infection, Two-way ANOVA, P = 0.97 and P = 0.13).


Figure 2
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Adjunctive treatment with anti-pneumococcal antiserum did not alter the development of (a) clinical scores in the acute disease stage or following recovery phase (P = 0.97). Similarly, motor performance scores (b) were unaffected by the administration of anti-pneumococcal antiserum (P = 0.13). The solid vertical arrow indicates time for initiation of treatments (26 h after infection).

 
Bacterial counts in CSF and blood samples obtained prior to treatment were equal between rats randomized to APAS or antibiotics only (Mann–Whitney, P = 0.80 and P = 0.71). Also CSF- and blood WBC counts were equal between rats randomized to APAS or antibiotics only (P = 0.09 and P = 0.75).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
This study found a significant inhibitory effect of APAS on the development of pneumococcal meningitis when administered at the time of infection. However, when APAS was administered as adjunctive therapy at the same time as antibiotic therapy, both outcome and disease scores were unaffected.

Since active immunization8 and, in the present study, passive immunization induced by pretreatment with APAS were incapable of controlling the local meningeal infection and inflammatory response, it is thought that the beneficial effect of APAS pretreatment is probably due to the eradication of pneumococci from the blood. This is supported by our own studies showing that the degree of bacteraemia, prior to initiation of antibiotic therapy, is associated with outcome in an experimental model of pneumococcal meningitis.7,9 When APAS was administered as adjunctive therapy 26 h after infection, the lack of efficacy could imply that at late disease stages, it is not the pneumococcal bacteraemia by itself that determines outcome. This suggests that the degree of bacteraemia may simply reflect disease severity e.g. the sepsis syndrome.

The present study design provides no mechanistic information relating to the lack of APAS efficacy and further evaluation of for example phagocytosis was not performed since concomitant administration of antibiotics efficiently reduces bacterial counts and sterilizes CSF and blood within 10–12 h after administration.

The presented results do not conflict with the work by Yuste et al.6 who showed that combined therapy with antibiotics and APAS (compared with monotherapy with antibiotics) reduced mortality in a pneumococcal sepsis model. Since Yuste et al. administered antibiotics and APAS prior to, or within 1 h after bacterial inoculation, the study resembles our pretreatment study and not the late adjunctive aspect of our work.

This study emphasizes the lessons learned in the pre-antibiotic era, where monotherapy with anti-pneumococcal antiserum had a beneficial effect on the outcome from pneumococcal pneumonia and bacteraemia5 but not from pneumococcal meningitis.10 Our data do not exclude the possibility that combined treatment with APASs and antibiotics could have a beneficial role if treatment were initiated earlier in the course of meningitis. However, modern antibiotics are capable of sterilizing the CSF and blood quickly and efficiently. Consequently, the need for additional infection control by anti-pneumococcal sero-therapy may be limited.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
Statens Serum Institut produces and manufactures pneumococcal serotype-specific antiserum for diagnostic purposes (Pneumosera).


   Acknowledgements
 
Financial support from the following foundations is gratefully acknowledged: Dagmar Marshalls Fond; Eivind Eckbos dansk-norske Legat; Lily Benthine Lunds Fond; Lundbeck Fonden; Direktør Jacob Madsen og Hustru Olga Madsens Fond and the SSAC foundation (Scandinavian Society for Antimicrobial Chemotherapy). The authors would like to thank Dorthe Truelsen and Jacob Vang for their expert technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 Ostergaard C, Konradsen HB, Samuelsson S. (2005) Clinical presentation and prognostic factors of Streptococcus pneumoniae meningitis according to the focus of infection. BMC Infect Dis 5:93.[CrossRef][Medline]

2 Weisfelt M, van de Beek D, Spanjaard L, et al. (2006) Clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series. Lancet Neurol 5:123–9.[CrossRef][Web of Science][Medline]

3 Kastenbauer S and Pfister HW. (2003) Pneumococcal meningitis in adults: spectrum of complications and prognostic factors in a series of 87 cases. Brain 126:1015–25.[Abstract/Free Full Text]

4 van de Beek D and de Gans J. (2004) Dexamethasone and pneumococcal meningitis. Ann Intern Med 141:327.[Free Full Text]

5 Avery OT, Chickering HT, Cole R, et al. (1917) Acute lobar pneumoniae. Prevention and serum treatment. Rockefeller Institute for Medical Research, no. 7.

6 Yuste J, Fenoll A, Casal J, et al. (2002) Combined effect of specific antibodies (as serotherapy or preimmunization) and amoxicillin doses in treatment of Streptococcus pneumoniae sepsis in a mouse model. Antimicrob Agents Chemother 46:4043–4.[Free Full Text]

7 Brandt CT, Lundgren JD, Lund SP, et al. (2004) Attenuation of the bacterial load in blood by pretreatment with granulocyte-colony-stimulating factor protects rats from fatal outcome and brain damage during Streptococcus pneumoniae meningitis. Infect Immun 72:4647–53.[Abstract/Free Full Text]

8 Ostergaard C, O'Reilly T, Brandt C, et al. (2006) The Influence of the blood bacterial load on the meningeal inflammatory response in Streptococcus pneumoniae meningitis. BMC Infect Dis 6:78.[CrossRef][Medline]

9 Brandt CT, Lundgren JD, Frimodt-Moller N, et al. (2005) Blocking of leukocyte accumulation in the cerebrospinal fluid augments bacteremia and increases lethality in experimental pneumococcal meningitis. J Neuroimmunol 166:126–31.[CrossRef][Web of Science][Medline]

10 Kolmer JA. (1929) Pneumococcus and streptococcus meningitis: chemotherapy and serumtherapy, with special reference to newer methods. JAMA 92:874.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/6/1291    most recent
dkl401v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Brandt, C. T.
Right arrow Articles by Østergaard, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandt, C. T.
Right arrow Articles by Østergaard, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?