a Laboratory of Experimental Infection, Infectious Diseases Service; b Microbiology Service, Ciutat Sanitària i Universitària de Bellvitge, C/Feixa Llarga s/n, 08907 L'Hospitalet, Barcelona, Spain
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Abstract |
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Introduction |
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Materials and methods |
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Serial ten-fold dilutions were performed to determine bacterial counts at each time point (the detection limit with this method was 102 cfu/mL, and a value of 0 (log 10 = 1) was assigned to the first and subsequent sterile cultures).
MICs of penicillin and ceftriaxone were determined by a microdilution method with cation-adjusted MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 3% lysed horse blood, with an inoculum of approximately 5 x 105 cfu/mL as recommended by the NCCLS.14 The MIC was defined as the lowest concentration of antibiotic for which no visible turbidity was apparent.
CSF ceftriaxone concentrations were determined by an agar disc diffusion method15 in antibiotic medium 2 (Difco Laboratories), using Bacillus subtillis ATCC 12432 as the indicator strain. Standard antibiotic solutions were prepared in saline. To avoid inter-day variability the concentrations of drugs from which a standard curve was determined and all CSF samples were assayed in duplicate in a single day. The assay variability for individual samples was 10%. The level of detection was 0.06 mg/L.
CSF bactericidal activities were performed by a microdilution method16 with cation-adjusted MuellerHinton broth supplemented with 25% lysed horse blood. Serial two-fold dilutions (range 1:2 to 1:4096) of CSF were performed, and a concentration of 5 x 105 cfu/mL of the same strain as the meningitis model was used for the inoculum. Bacteriostatic activity was defined as the highest dilution without visible turbidity and bactericidal activity was defined as the highest dilution capable of killing 99.9% of the initial inoculum.
To minimize the effect of carry over of antibiotic agent, an entire agar plate was used for each sample. The sample was placed on the plate in a single streak down the centre, allowed to be absorbed into the agar until the plate surface appeared dry, and then the inoculum was spread over the plate.17
Therapeutic failure was defined as an increase in bacterial concentration compared with a previous count, and the death of the rabbit.
Since slight but not statistically significant differences in CSF ceftriaxone concentrations were found at certain time points, it was decided to perform a further set of experiments in order to determine the area under the timeconcentration curve (AUC; by the linear trapezoidal rule), which might better demonstrate such differences. The rabbit model was set up as before and 13 rabbits were treated with ceftriaxone 100 mg/kg/day and 13 rabbits with ceftriaxone 100 mg/kg/day plus dexamethasone. Several samples of 50 µL of CSF were withdrawn at 1, 2, 4, 6, 8, 10, 12, 24, 26, 28, 30, 32, 34, 36 and 48 h. Only bacterial concentrations and CSF ceftriaxone concentrations were determined for these samples.
Statistical analysis
Fisher's exact test was used, when appropriate, to determine categorical variables. Student's t test was used to determine continuous variables. The N-par MannWhitney U Wilcoxon rank sum test was used to compare median bactericidal titres.
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Results |
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Ceftriaxone at 50 mg/kg/day reduced bacterial counts by >3 log cfu/mL at 24 h, with a mean log cfu/mL of 1.69 at 24 h versus 5.91 at 0 h (Figure). However, in two animals bacterial counts were above the level of detection at 24 h, and in one animal bacteria were detected at 48 h.
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Ceftriaxone at 100 mg/kg/day decreased bacterial counts by >3 log cfu/mL at 6 h, with a mean log cfu/mL 1.68 versus 5.6 at 0 h. With this regimen, at 24 h 5 of 8 animals had bacterial counts under the level of detection and at 48 h bacterial counts in all animals were under the level of detection. A higher bacterial count was found with concomitant use of dexamethasone, although the difference was not statistically significant: the mean log cfu/mL was 1.18 in ceftriaxone- treated animals versus 2.18 in ceftriaxone plus dexamethasone-treated animals at 24 h, and 0 in ceftriaxone-treated animals versus 1.2 mean log cfu/mL in ceftriaxone plus dexamethasone-treated animals at 48 h (Figure).
Comparisons between bacterial counts with the different regimens at a range of time points are shown in the Figure. Bacterial counts at 24 h in animals treated with ceftriaxone 100 mg/kg/day versus animals treated with ceftriaxone 50 mg/kg/day plus dexamethasone also showed statistically significant differences (P < 0.05).
CSF ceftriaxone concentrations
Mean CSF ceftriaxone concentrations at peak and trough on the first and second days of therapy are shown in Table I. For the 50 mg/kg/day dose these were lower when concomitant dexamethasone was used, but this difference was not statistically significant. Differences were more evident at 2 h (peak of the first day): 3.57 mg/L in the ceftriaxone group versus 1.75 mg/L in the ceftriaxone plus dexamethasone group (P = 0.06). Mean trough CSF ceftriaxone concentrations were below the MIC for the infecting strain at 24 and 48 h; the variations between groups were unremarkable.
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Area under the timeconcentration curve
No statistically significant differences were observed in CSF ceftriaxone concentrations between the time points during the first 24 h (Table II). Nor were statistically significant differences observed in the AUC of the two therapy groups: 25.81 mgh/L in the ceftriaxone group versus 23.61 mgh/L in the ceftriaxone plus dexamethasone group. After 24 h there was a higher mortality in the ceftriaxone plus dexamethasone group, and so comparisons of CSF levels and AUC calculations were not possible.
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This set of experiments also allowed the time above the MIC to be determined: on the first day this was 46 h (20%) in the ceftriaxone (with or without dexamethasone) groups and after the second dose it ranged 68 h (29%) in both groups.
CSF bactericidal activity
Median CSF bactericidal activities are shown in Table I. There were no statistically significant differences between groups, and all bacterial activities were low (
1:2). With the higher dose of ceftriaxone, bactericidal activities were also low, but were one dilution higher.
Therapeutic failures
The dose of 50 mg/kg/day resulted in therapeutic failures in 2/7 subjects (28%). Concomitant use of dexamethasone resulted in a higher failure rate: 4/7 (57%). The only group in which there were no therapeutic failures (0/8) was for those given ceftriaxone 100 mg/kg/day alone, but in the group given 100 mg/kg/day plus dexamethasone there were 2/7 (28%) therapeutic failures. The difference in therapeutic efficacy between the ceftriaxone 100 mg/kg/day and ceftriaxone 50 mg/kg/day plus dexamethasone groups was statistically significant (P < 0.05).
Inflammatory activity
Concomitant use of dexamethasone resulted in differences in CSF inflammatory activity. With the dose of 50 mg/kg/ day the mean CSF WBC at 24 h was 11,900 cells/µL in the ceftriaxone group versus 4780 cells/µL in the ceftriaxone plus dexamethasone group (P < 0.05). With ceftriaxone 100 mg/kg/day no differences were observed in the mean WBC between the groups treated with or without dexamethasone, but there were statistically significant differences in the mean CSF lactic acid concentration at 48 h: 6.71 mg/L in the ceftriaxone group versus 3.18 mg/L in the ceftriaxone plus dexamethasone group (P < 0.05).
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Discussion |
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Adult patients with pneumococcal meningitis cannot usually be treated with 100 mg/kg/day of ceftriaxone, because of the limitation in total daily dose (4 g) recommended by the manufacturers. It is possible to increase the dose of other similar broad-spectrum cephalosporins, such as cefotaxime, for which a total daily dose limitation is not given. Our group has reported the successful use of cefotaxime 300 mg/kg/day concomitantly with dexamethasone in ten cases of pneumococcal meningitis caused by strains with decreased susceptibility to third-generation cephalosporins.6 Increased dosage of third-generation cephalosporins might be an effective therapy for cephalosporin-resistant pneumococcal meningitis, at least for MICs
2 mg/L. However, in our study, with the use of dexamethasone the results were unacceptable even at the higher recommended dose. We have previously shown11 that dexamethasone can be used safely with ceftriaxone to treat penicillin-sensitive strains. In this study, although small differences were detected, there were no statistically significant differences in ceftriaxone levels in the CSF, bactericidal activities or therapeutic failures. Paris et al.12 found that ceftriaxone concentrations were lower at each time point in animals given dexamethasone in a meningitis model using two cephalosporin-resistant pneumococcal strains, although again the differences were not statistically significant. These small, though not statistically significant differences might be of biological relevance in cases owing to cephalosporin-resistant pneumococci. In the present study, AUC data showed that dexamethasone did not decrease ceftriaxone levels in the CSF. However, concomitant use of dexamethasone worsened results in terms of bacterial killing and bactericidal activity, with a higher number of therapeutic failures for both dose regimens. The mechanism of these failures remains unclear. Although possible differences in CSF ceftriaxone concentrations may appear the best explanation, this is not supported by our data. Nevertheless, percentage of time above MICs of the dosing intervals was lower than accepted for achieving therapeutic success in most cases (therapeutic failures with percentage of time above the MIC values in the range of 30% might be expected).19 The concomitant use of dexamethasone may accentuate this therapeutic limitation. Our data indicate that increasing doses of ceftriaxone may be an effective mode of therapy for cephalosporin-resistant cases, but concomitant use of dexamethasone makes this approach unreliable.
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Acknowledgments |
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Notes |
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References |
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Received 25 May 1999; returned 9 August 1999; revised 24 September 1999; accepted 12 November 1999