Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA
Received 11 January 2003; returned 24 February 2003; revised 14 July 2003; accepted 15 July 2003
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Abstract |
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Keywords: animal models, CSF, experimental meningitis, inflammatory response, S. pneumoniae
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Introduction |
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Currently, dexamethasone (DXM) is the only anti-inflammatory agent that has been documented to improve the outcome of bacterial meningitis in clinical trials, most convincingly in children with Haemophilus influenzae meningitis.6 DXM inhibits production of TNF- and IL-1, reverses development of brain oedema and limits the increase in cerebrospinal fluid (CSF) lactate and leucocyte concentrations.1,7,8 Studies have suggested that in H. influenzae meningitis the timing of DXM in relation to the first antibiotic injection is critical. The antibiotic-induced inflammatory response is prevented only if DXM therapy is given simultaneously with ceftriaxone; DXM given 1 h later failed to modulate the antibiotic-induced inflammatory response.3 A meta-analysis of 10 clinical trials in children suggested that the timing of DXM therapy might also be critical in pneumococcal meningitis. DXM therapy prevented the development of severe hearing loss only when it was given before or at the same time as the first antibiotic injection (OR = 0.09; 95% CI: 0.000.71).6 Recently de Gans & van de Beek9 in a prospective, randomized, placebo-controlled, multicentre trial demonstrated the beneficial effect of adjunctive DXM therapy on the outcome of pneumococcal meningitis in adults.
The present study was conducted to evaluate the modulatory effect of DXM therapy given 30 min before, compared with 1 h after, intracisternal injection of pneumococcal cell walls, or after the first injection of ampicillin, in experimental meningitis caused by live pneumococci.
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Material and methods |
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Pneumococcal cell wall (1 cm3 of this product was obtained from 2.5 x 109 live organisms) was produced and provided by Dr Elaine Tuomanen.10 Streptococcus pneumoniae (MIC and MBC of ampicillin, 0.01 mg/L) was originally isolated from a patient with bacterial meningitis. To induce meningitis, 0.2 mL pneumococcal cell wall product, or 104105 cfu/mL of live organisms, were inoculated intracisternally.
Meningitis model and treatment
A rabbit meningitis model originally described by Dacey & Sande11 was used. In the first set of experiments, DXM (1 mg/kg) was given intravenously 30 min before or 1 h after the administration of pneumococcal cell walls to six and seven animals, respectively. In the second set of experiments, 1618 h after inoculation of live organisms, animals were treated with ampicillin alone (75 mg/kg every 12 h) for 24 h, or with the combination of ampicillin and intravenous DXM (1 mg/kg) given 30 min before or 1 h after the first ampicillin dose. Each treatment group consisted of 1213 animals. No treatment was given to control animals.
CSF sample collection and analysis
CSF was collected directly from the cisterna magna under acepromazine and ketamine anaesthesia before and 2, 4, 6, 12 and 24 h after start of therapy. For the first four CSF collections, animals were restrained under anaesthesia in stereotactic frames. Leucocytes were counted in a Neubauer haemocytometer. Bacterial concentrations in CSF were measured by plating undiluted and serial dilutions of CSF on sheep blood agar and incubating in 5% CO2 at 35°C for 24 h. The lower limit of detection was 10 cfu/mL. The remaining CSF was centrifuged and the supernatant stored at 70°C. CSF lactate concentrations were measured by a photocolorimetric assay (Behring Diagnostics Inc, Milton Keynes, UK). TNF- concentrations were measured by cytotoxic assay using L929 tumorigenic murine fibroblasts.12 The standard curve for the TNF-
assay was linear from 402500 pg/mL.
Statistical analysis
Normally distributed data are presented as mean ± S.D. and non-normally distributed data as median and range. Students t-test was used for comparison of parametric data and the KruskallWallis analysis of variance for non-parametric variables.
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Results |
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Administration of pneumococcal cell walls resulted in the release of TNF-, an influx of leucocytes and increased lactate concentrations in CSF (Figure 1). The elevation in TNF-
concentrations was prevented by DXM therapy when given 30 min before or 1 h after pneumococcal cell walls. The increase in CSF leucocyte and lactate concentrations, however, was inhibited only when DXM therapy was given 30 min before the cell wall products.
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The addition of DXM to ampicillin therapy resulted in a lower initial bacterial killing rate compared with ampicillin therapy alone (0.39 ± 0.1 cfu/mL/h versus 0.57 ± 0.12 cfu/mL/h; P = 0.04). The changes in CSF inflammatory indices were similar in all study groups and were not influenced by the co-administration of DXM (Figure 2).
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Discussion |
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Liberation of free endotoxin or cell-wall components by cell-wall active antibiotics has been demonstrated in vitro and in experimental meningitis.3,4,5,13 This is associated with enhanced inflammation in the subarachnoid space, as evidenced by an increase in leucocyte, TNF- and lactate concentrations.3,14 Our study showed that the antibiotic-induced inflammatory burst did not occur in all animals and was seen only in those with greater CSF bacterial concentrations (
5.6 log10 cfu/mL). Although this has been intimated previously, data were not provided.14 In experimental meningitis, the magnitude of the inflammatory response in CSF depends on the concentration of inoculated cell walls10 and thus it is not surprising that animals with higher bacterial counts demonstrate a more pronounced host inflammatory response after antibiotic therapy.
There is concern that rapid bacterial killing by antibiotics could result in an enhanced inflammatory response and a worsening of the clinical outcome in meningitis.15,16 The results of this and previous studies, however, do not support these speculations. On the contrary, clinical and experimental studies have demonstrated that rapid effective bactericidal therapy protects against the development of deafness and other neurological disabilities.17,18 Moreover, this and previous studies4 found no correlation between CSF inflammatory markers and the magnitude of bacterial killing. Uncontrolled bacterial growth eventually results in a much greater release of cell wall componentsand consequent enhancement of the inflammatory responsethan that induced by antibacterial therapy.4,13 These findings suggest that early antibacterial therapy and rapid clearance of bacteria from CSF outweigh the potential adverse effects caused by the antibiotic-induced inflammatory burst.
DXM, as an adjunct to antibacterial therapy in bacterial meningitis, has been shown to be beneficial in H. influenzae meningitis in children.6 In pneumococcal meningitis, however, its modulatory effect was uncertain because of the relatively small numbers of children enrolled in prospective controlled trials.19 In adults with pneumococcal meningitis, an unfavourable outcome was seen in 26% of patients receiving adjunctive DXM compared with 52% among those receiving placebo.9 The results of our study clearly supported previous findings by Tuomanen et al.8 and showed that, similar to H. influenzae meningitis, DXM therapy prevents the antibiotic-induced release of TNF- and lactate concentrations in CSF in pneumococcal meningitis.3
Early institution of DXM therapy has been suggested because of its delayed onset of action.20 In our study, the timing of DXM appeared to be critical only in meningitis induced by the pneumococcal cell wall. In meningitis induced by live microorganisms, however, once CSF inflammation was established, administration of DXM before the dose of ampicillin did not appear to have a significantly greater salutary effect than 1 h after ampicillin; DXM administration that was delayed further was not assessed. These results contrast with those obtained in experimental H. influenzae meningitis, where the antibiotic-induced inflammatory response was modulated only if DXM was given before or simultaneously with ceftriaxone therapy.3 The effectiveness of early DXM administration was also highlighted in a recent meta-analysis of 10 randomized controlled clinical trials.6
At the time of diagnosis, 40% of patients with pneumococcal meningitis have CSF bacterial concentrations <106 cfu/mL21, and our findings suggest that antibiotic-induced enhanced inflammation may not occur in such patients. Whether these patients benefit from DXM therapy, and how they can be identified at diagnosis, requires further clarification. Detection of bacteria in Gram-stained specimens of CSF may be useful in identifying patients with large bacterial loads.22
One possible detrimental effect of DXM therapy, also demonstrated in this study, is decreased bacterial clearance from CSF.23,24 DXM decreases the permeability of the bloodbrain barrier, resulting in decreased concentrations of hydrophilic antibiotics in the CSF.25 Also, high concentrations of DXM (400 µg/mL) inhibit phagocytosis by CSF leucocytes.26The clinical relevance of these findings, however, has not been demonstrated.6,9
In this model of pneumococcal meningitis, CSF bacterial concentrations at the start of therapy appeared to be more important than the timing of DXM therapy in influencing the antibiotic-induced inflammatory response. It is likely that there is a time beyond which DXM loses its effectiveness, but this point has not been clearly defined.
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Acknowledgements |
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Footnotes |
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References |
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