Experimental study of the efficacy of vancomycin, rifampicin and dexamethasone in the therapy of pneumococcal meningitis

J. Martínez-Lacasaa, C. Cabellosa,*, A. Martos, A. Fernándeza, F. Tubaub, P. F. Viladricha, J. Liñaresb and F. Gudiola

a Laboratory of Experimental Infection, Infectious Diseases Service and b Microbiology Service, Ciutat Sanitària i Universitaria de Bellvitge, Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The object of the study was to assess the efficacy of rifampicin and the combination of rifampicin plus vancomycin in a rabbit model of experimental penicillin-resistant pneumococcal meningitis. We also studied the effect of concomitant dexamethasone on the CSF antibiotic levels and inflammatory parameters. The rabbit model of pneumococcal meningitis was used. Groups of eight rabbits were inoculated with 106 cfu/mL of a cephalosporin-resistant pneumococcal strain (MIC of cefotaxime/ceftriaxone 2 mg/L). Eighteen hours later they were treated with rifampicin 15 mg/kg/day, vancomycin 30 mg/kg/day or both ± dexamethasone (0.25 mg/kg/day) for 48 h. Serial CSF samples were withdrawn to carry out bacterial counts, antibiotic concentration and inflammatory parameters. Rifampicin and vancomycin promoted a reduction of >3 log cfu/mL at 6 and 24 h, and cfu were below the level of detection at 48 h. Combination therapy with vancomycin plus rifampicin was not synergic but it had similar efficacy to either antibiotic alone and it was able to reduce bacterial concentration below the level of detection at 48 h. Concomitant use of dexamethasone decreased vancomycin levels when it was used alone (P< 0.05), but not when it was used in combination with rifampicin. Rifampicin alone at 15 mg/kg/day produced a rapid bactericidal effect in this model of penicillin-resistant pneumococcal meningitis. The combination of vancomycin and rifampicin, although not synergic, proved to be equally effective. Using this combination in the clinical setting may allow rifampicin administration without emergence of resistance, and possibly concomitant dexamethasone administration without significant interference with CSF vancomycin levels.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The global increase in penicillin-resistant pneumococci has complicated the treatment of pneumococcal infections. The combination of third-generation cephalosporins and vancomycin is recommended for pneumococcal meningitis caused by resistant strains as well as for initial empirical therapy.1 In patients with penicillin allergy and in those infected by hypothetical strains with very high cephalosporin resistance, the use of non-ß-lactam antibiotics may be warranted. Chloramphenicol, the classical alternative approach for allergic patients, is not recommended nowadays because of the high frequency of resistance among pneumococci, ranging from 18 to 35%,2 and the poor bactericidal activity of the drug against these resistant strains.3 Monotherapy with systemic vancomycin in adults has been associated with clinical and bacteriological failures, best explained by the erratic and borderline vancomycin CSF levels, which become lower when concomitant dexamethasone is administered.4 Rifampicin has low MICs for most pneumococcal strains but, when studied by killing curves, is bacteriostatic rather than bactericidal and some antibiotic combinations seem to be antagonistic in vitro.5 Previous experimental studies have indicated that rifampicin is a less than optimal therapy for pneumococcal meningitis, due to its slow bactericidal activity, inferior to that of ceftriaxone, vancomycin and ofloxacin;6 in one experiment, the dose– response in vivo showed a paradoxical reduction in activity at very high levels.6 In addition, the use of rifampicin as monotherapy has been associated with the rapid emergence of resistance in the course of treatment.7 New fluoroquinolones may be a promising alternative but there is not enough experimental or clinical experience. In this scenario, the efficacy of different antibiotic combinations should be further investigated.

The aim of the present study was to assess the efficacy of the combination rifampicin plus vancomycin in a rabbit model of experimental pneumococcal-resistant meningitis. We also studied the effect of concomitant dexamethasone on the CSF antibiotic levels and inflammatory parameters.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strain

A Streptococcus pneumoniae strain recovered from a patient with pneumococcal meningitis was used. The strain, named 2349, belonged to serotype 23. MICs were determined by the microdilution method in cation-supplemented Mueller– Hinton broth with 5% whole defibrinated horse blood and the appropriate concentration of antibiotic. The wells of microdilution plates were inoculated to a volume of 100 µL with an inoculum containing 106 cfu/mL. The MIC was defined as the lowest concentration of antibiotic that prevented growth visible without the microscope after an overnight incubation of plates at 35°C. MICs were as follows: penicillin 4 mg/L; ceftriaxone/cefotaxime 2 mg/L; vancomycin 0.25 mg/L; rifampicin 0.03 mg/L. In vitro killing curves with this strain have been published previously.8

Rabbit model

The rabbit model of meningitis was carried out according to an established protocol.9 The study was approved by the Ethical Committee for Animal Experiments at the University of Barcelona (Campus de Bellvitge). For all experiments, rabbits were challenged in groups of at least eight animals. Two kilogram female New Zealand white rabbits were anaesthetized with ketamine 35 mg im (Ketolar; Parke-Davis, El Prat de Llobregat, Spain) and xylazine 5 mg (Rompun; Bayer AG, Leverkusen, Germany) per kg of body weight, and a dental acrylic helmet was affixed to the calvarium; 24 h later, the animals were anaesthetized again and placed in a stereotaxic frame. A spinal needle was introduced into the cisterna magna, 200 µL of CSF was withdrawn and 200 µL of 106 cfu/mL of the pneumococcal strain were inoculated. Eighteen hours later the rabbits were anaesthetized again with urethane (1.75 g/kg, sc) (Sigma Chemical Company, St Louis, MO, USA) and thiopental 5 mg/g, iv (Penthotal sodico; Abbott Laboratories, Madrid, Spain), and a baseline CSF sample was taken. Then an iv dose of dexamethasone (Fortecortin; Merck, Mollet del Vallés, Barcelona, Spain) or saline (Suero fisiológico Braun; Braun S.A. Rubí, Barcelona, Spain) was administered. The total dose was 0.5 mg/24 h divided every 12 h over a 48 h period (four doses). Ten minutes later iv antibiotics were administered at standard doses: rifampicin (Rifaldin; Hoechst-Marion-Roussell, Barcelona, Spain), 15 mg/kg/24 h in a single daily dose (two doses); vancomycin (Diatracin; Lilly SA, Alcobendas, Spain), 30 mg/kg/ 24 h divided every 12 h (four doses). Therapeutic groups were as follows: rifampicin, vancomycin, rifampicin plus vancomycin with and without dexamethasone and control.

Serial CSF samples were taken at 2 (peak), 4, 6, 12, 24 (trough), 26 (peak) and 48 (trough) h of treatment. CSF samples were used to carry out direct bacterial cultures, vancomycin and rifampicin peak and trough CSF levels and trough and peak CSF bactericidal activities. Serial 10-fold dilutions were made to determine bacterial counts at each time point (detection limit 102 cfu/mL), a value of 1.9 log cfu/mL was assigned to the first sterile culture and of 0 to the subsequent ones. To avoid carryover antimicrobial agent interference, an entire agar plate was used for each sample. The sample was placed on to 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.10 Vancomycin levels were determined by fluorescence polarization immunoassay with a limit of detection of 0.06 mg/L. Rifampicin levels were analysed by high performance liquid chromatography with a limit of detection of 0.05 mg/L. CSF bactericidal activities were carried out by a microdilution method with cation-adjusted Mueller–Hinton broth supplemented with 5% lysed horse blood. Serial two-fold dilution (range 1:2 to 1:4096) of CSF samples was carried out, and a concentration of 5 x 105 cfu/mL of the same strain used in the meningitis model was inoculated into each well. After inoculation at 35°C for 24 h, bacteriostatic activity was read as the highest dilution without turbidity. One hundred microlitres of each well without turbidity was subcultured at 35°C for 24 h and bactericidal activity was read as the lowest concentration of antimicrobial agent capable of killing 99.9% of the inoculated bacteria. For leucocyte counts, 10 µL of each sample were diluted 1:1 with Turk solution (made in-house) and read with a Neubauer chamber. After centrifugation, CSF was stored at -80°C in order to determine the remaining inflammatory parameters. CSF protein concentration was determined by Bradford's method (Bio-Rad Protein Assay), and CSF lactate concentration with Lactate PAP (bioMérieux SA, Marcy l'Étoile, France). Brain oedema was determined by comparing wet weight (tested immediately after extraction) and dry weight (after 7 days in a 100°C stove). The difference is expressed as grams of water/100 g dry weight and oedema is defined above 400.

Bactericidal effect was defined as a decrease of >=3 logs cfu/mL. Therapeutic failure was defined as an increase in bacterial concentration compared with a previous count during therapy with death of the rabbit. For statistical analysis, a {chi}2 test or Fisher exact test was used when appropriate to determine categorical variables. The N-par Mann– Whitney U Wilcoxon rank sum test was used to compare median bactericidal activities.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Curves representing bacterial concentrations obtained at each time point (0, 2, 6, 24 and 48 h) with the different therapeutic regimens are shown in the Figure. CSF antibiotic levels, bactericidal titres and logarithmic reduction in bacterial counts at 6 and 24 h with the different therapeutic regimens are shown in Table 1Go. Inflammatory parameters and brain oedema values are shown in Table 2Go


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Table 1. Mean CSF rifampicin and vancomycin level ± s.d. (mg/L) and mean decrease in bacterial counts ({triangleup}log cfu/mL ) in rabbits infected with strain 2349 of S. pneumoniae
 

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Table 2. Inflammatory activity in CSF of rabbits infected with strain 2349 of S. pneumoniae
 
Vancomycin

Vancomycin was almost bactericidal at 6 h, with a mean bacterial concentration of 2.89 ± 0.72 cfu/mL, whereas the mean initial bacterial concentration was 5.75 ± 0.98 cfu/ mL. At 24 h the bacterial concentration was 1.63 ± 0.66 and at 48 h under the limit of detection in all cases and there were no therapeutic failures.

Mean CSF vancomycin levels at 2 and 26 h (peak) were 1.89 ± 1.01 and 1.76 ± 0.58 mg/L; at 24 and 48 h (trough) they were 0.70 ± 0.40 and 0.56 ± 0.50 mg/L.

Median CSF bactericidal activities at 2 and 26 h (peak) were 1:4; and at 24 and 48 h (trough) were 1:2.

With vancomycin and dexamethasone in combination the mean bacterial concentration at 6 h was 2.85 ± 0.80 cfu/mL, whereas the mean initial bacterial concentration was 5.09 ± 0.56 cfu/mL. At 24 h the mean bacterial concentration was 1.59 ± 0.71 cfu/mL and at 48 h it was 0.27 ± 0.6 cfu/mL. There were no therapeutic failures.

Mean CSF vancomycin levels were significantly lower in animals treated with dexamethasone than in the group treated with vancomycin alone. The mean value at 2 h was 0.83 ± 0.70 mg/L and at 26 h 0.70 ± 0.94 mg/L (P < 0.05 compared with mean vancomycin concentration in vancomycin treated animals). Brain oedema was significantly lower in the vancomycin–dexamethasone group than in the vancomycin group, with a mean value of 382.6 ± 35 versus 417.2 ± 29 g water/100 g dry weight (P < 0.05). Other inflammatory parameters are shown in Table 2Go.

Rifampicin

Rifampicin achieved a bactericidal effect at 6 h, with a mean bacterial concentration of 2.91 ± 0.98 log cfu/mL, whereas the initial concentration was 5.91 ± 0.58 log cfu/mL. At 24 h the mean bacterial concentration was 1.90 ± 0 log cfu/mL and below the limit of detection at 48 h. There were no therapeutic failures.

Mean CSF rifampicin levels at 2 h (peak) were 0.45 ± 0.26 mg/L, and at 12 h (trough) 0.16 ± 0.08 mg/L.

Median CSF bactericidal activities at 2 and 26 h (peak) were 1:8–1:16; and at 24 and 48 h (trough) were 1:2–1:4. Brain oedema showed a mean value of 406 ± 32 g water/ 100 g dry weight.

Rifampicin plus vancomycin

The association of rifampicin plus vancomycin did not quite achieve a bactericidal effect at 6 h but it was close. At this time point the mean bacterial concentration was 2.95 ± 1.41 log cfu/mL, whereas the initial bacterial concentration was 5.55 ± 0.89 cfu/mL. At 24 h there was a marked bactericidal effect, with a mean bacterial concentration of 1.67 ± 0.63 log cfu/mL. At 48 h bacterial concentration was below the level of detection. There were no therapeutic failures.

Mean CSF vancomycin levels at 2 and 26 h (peak) were 1.13 ± 0.96 and 1.74 ± 0.54 mg/L; at 24 and 48 h (trough) they were 0.28 ± 0.32 and 0.71 ± 0.10 mg/L. Mean CSF rifampicin level at 2 h was 0.42 ± 0.38 mg/L.

Median CSF bactericidal activities at 2 and 26 h (peak) were 1:4–1:8; at 24 and 48 h (trough) were 1:2–1:4. Brain oedema showed a mean value of 414.17 ± 39.46 g water/ 100 g dry weight.

The addition of dexamethasone to the combination of rifampicin plus vancomycin did not produce significant variations in mean bacterial concentrations. At 6 h it was 3.58 ± 0.8 log cfu/mL, at 24 h it was 1.96 ± 0.13 log cfu/mL and at 48 h it was below the level of detection. There were no therapeutic failures.

Mean CSF vancomycin levels at 2 h were similar to those obtained in the group without dexamethasone, with a mean value of 1.34 ± 0.44 mg/L, but at 26 h they were lower, without statistically significant differences, with a mean value of 0.64 ± 0.43 mg/L. Mean brain oedema was significantly lower than in the group without dexamethasone, 391 ± 32 versus 414 ± 39 g water/100 g dry weight (P < 0.05). Other inflammatory parameters showed a trend to lower values in the dexamethasone group but without statistical significance (Table 2Go).

Control group

Mean bacterial concentration increased in the first hour from 6.36 ± 0.70 log cfu/mL as initial count (18 h after inoculation and 0 h of therapy in the therapeutic groups) up to 6.75 ± 1.25 log cfu/mL at 24 h and it was slightly lower in the following count.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Results obtained with vancomycin alone, including CSF levels, bactericidal activity and therapeutic efficacy, were all in accordance with previous experiments carried out in the rabbit meningitis model.9 CSF vancomycin peak levels ranged between four and six times the MIC for this particular penicillin-resistant pneumococcal strain; the drug was bactericidal between 6 and 24 h and there were no therapeutic failures. Likewise, our data reinforce the concept that concomitant dexamethasone administration lowers CSF vancomycin levels, as has been reported.11,12 However, some variability of vancomycin concentration between groups may be ascribed in part to its erratic CSF penetration. In our experiment, this decrease in CSF vancomycin levels was associated with a slower bactericidal activity.

Rifampicin showed good bactericidal activity, similar to that of vancomycin, achieving a bactericidal effect at 6 h and a comparable efficacy.

These results are in contrast to those found earlier by Nau et al.,6 who reported slow bactericidal activity when using rifampicin in the same model. However, they administered rifampicin (10 mg/kg) by continuous infusion for 7 h, and measured reduction of bacterial concentration at this time point, whereas we used a single daily dose of 15 mg/kg (two doses) and measured reduction of bacterial concentration at 2, 6, 24 and 48 h. They obtained a median CSF rifampicin concentration of 4.6 ± 1.7 mg/L at 7 h (microbiological method) and we obtained a value of 0.45 ± 0.16 mg/L at 2 h (HPLC). These major differences may account for the discrepancies between the two studies. In fact, Nau et al. emphasized the occurrence of a paradoxical dose–response effect in their experiment. Maybe achieving a lower rifampicin CSF concentration, closer to that seen in humans during rifampicin therapy at conventional dosage,13 is more convenient in order to obtain a better bactericidal activity.

In a recent study of pneumococcal meningitis in mice, Nau et al.14 have shown that rifampicin-treated animals had lower CSF concentrations of lipoteichoic and teichoic acid than ceftriaxone-treated mice and also a lower mortality rate, indicating that rifampicin has a lower potential for enhancing CSF inflammatory response. Our experiments did not show statistically significant differences between rifampicin and vancomycin regarding other CSF inflammatory parameters like WBC, lactate and protein concentration.

Emergence of resistance to rifampicin did not occur in this study; however, we have observed this well-known phenomenon when carrying out killing curves with several pneumococcal strains and also in vivo using a serotype 3, penicillin-susceptible pneumococcal strain in the rabbit model (data not shown). This fact argues against the use of rifampicin as monotherapy, whatever the intensity of its bactericidal activity against pneumococci. On the other hand, our own data and other experiences indicate that emergence of rifampicin resistance during therapy does not occur when administering it as part of combination therapy.

The combination of rifampicin and vancomycin produced a bactericidal activity similar to that observed with either antibiotic alone, without any additive or synergic effect. An antagonistic effect has been described in some in vitro studies,5,8 but this effect was not present when our in vivo experiments were carried out. The bactericidal activities in the peak ranging from 1:8 to 1:16 seem adequate to produce the sterilization of CSF, which was observed at 48 h in all cases. It is noteworthy that the interference of dexamethasone on the CSF vancomycin levels appeared to be less pronounced when rifampicin was used simultaneously. This fact might be explained by an interaction between rifampicin and dexamethasone, resulting in an enhanced dexamethasone metabolism and subsequent lower dexamethasone levels. This interaction is well known with several drugs including corticosteroids and other antibiotics. A decrease in steroid concentration has been demonstrated in several clinical situations and rifampicin has been able to precipitate Addisonian crisis in patients with substitutive therapy for adrenal insufficiency. Also rifampicin has been able to decrease doxycycline levels in brucellosis therapy and co-trimoxazole levels in AIDS patients with PCP pneumonia.15–17 Nevertheless, in our experiments, a marked reduction in brain oedema was observed in all groups receiving dexamethasone, indicating a beneficial effect on the inflammatory response even in the presence of rifampicin.

In conclusion, rifampicin alone was effective in the therapy of pneumococcal meningitis. The combination of vancomycin and rifampicin, although not synergic, produced a rapid bactericidal effect and proved to be effective in this model of penicillin-resistant pneumococcal meningitis. Using this combination in the clinical setting may both allow rifampicin administration without emergence of resistance, and concomitant dexamethasone administration without significant interference with CSF vancomycin levels. It may constitute a good alternative to ß-lactam therapy, which could be evaluated in humans.



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Figure. Bacterial concentration in CSF with several antibiotic regimens. Key to symbols: x, vancomycin; {blacktriangleup}, vancomycin plus dexamethasone; {diamondsuit}, rifampicin; *, vancomycin plus rifampicin; {square}, vancomycin plus rifampicin plus dexamethasone; {diamondsuit} , control.

 

    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by grants 92/277 and 93/246 from the ‘Fondo de Investigaciones Sanitarias de la Seguridad Social’ (FIS), National Health Service, Spain. J. Martínez-Lacasa, A. Martos, F. Tubau and A. Fernández were supported by grants from the ‘Fundació August Pi i Sunyer’.


    Notes
 
* Correspondence address. Infectious Diseases Service, Hospital de Bellvitge, C/Feixa Llarga s/n, 08907 L'Hospitalet, Barcelona, Spain. Tel: +34-93-3357011 ext. 2487; Fax: +34-93-2607637; E-mail: ccabellos{at}csub.scs.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Friedland, I. R., Paris, M., Ehrett, S., Hickey, H., Olsen, K. & McCracken, G. H., Jr (1993). Evaluation of antimicrobial regimens for experimental penicillin and cephalosporin resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 37, 1630–6.[Abstract]

2 . Liñares, J., Tubau, F. & Domínguez, M. A. (2000). Antibiotic resistance in Streptococcus pneumoniae in Spain: an overview of the 1990s. In Streptococcus Pneumoniae, Molecular Biology and Mechanisms of Disease, 1st edn, (Tomasz, A., Ed.), pp. 399–407. Mary Ann Liebert Inc., Larchmont, NY.

3 . Friedland, I. R. & Klugman, K. P. (1992). Failure of chloramphenicol therapy in penicillin-resistant pneumococcal meningitis. Lancet 339, 405–8.[ISI][Medline]

4 . Viladrich, P., Gudiol, F., Liñares, J., Rufí, G., Ariza, X. & Pallarés, R. (1991). Evaluation of vancomycin in the therapy of adult pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 35, 2467–72.[ISI][Medline]

5 . Doit, C. P., Bonacorsi, S. P., Fremaux, A. J., Sissia, G., Cohen, R., Geslin, P. L. et al. (1994). In vitro activities of antibiotics at clinically achievable concentrations in cerebrospinal fluid against penicillin resistant Streptococcus pneumoniae isolated from children with meningitis. Antimicrobial Agents and Chemotherapy 38, 2655–9.[Abstract]

6 . Nau, R., Kaye, K., Sachdeva, M., Sande, E. R. & Tauber, M. G. (1994). Rifampicin for therapy of experimental pneumococcal meningitis in rabbits. Antimicrobial Agents and Chemotherapy 38, 1186–9.[Abstract]

7 . McCabe, W. R. & Lorian, V. (1968). Comparison of the antibacterial activity of rifampicin and other antibiotics. American Journal of Medical Science 256, 255–65.[ISI]

8 . Tubau, F., Liñares, J., Ardanuy, C., Martínez-Lacasa, J., Fernández, A., Cabellos, C. et al. (1996). Acticidad bactericida in vitro de diferentes combinaciones de antibióticos frente a Streptococcus pneumoniae resistente a penicilina y cefotaxima. Enfermedades Infecciosas y Microbiología Clínica 14, 590–5.

9 . Dacey, M. G. & Sande, M. A. (1974). Effect of probenecid on cerebrospinal fluid concentrations of penicillin and cephalosporin derivates. Antimicrobial Agents and Chemotherapy 57, 437–41.

10 . Knapp, C. & Moody, J. A. (1992). Test to assess bactericidal activity. In Clinical Microbiology Procedures Handbook, (Isenberg, H. D., Ed.), Section 5.16, p. 12. American Society for Microbiology, Washington, DC.

11 . Cabellos, C., Martínez-Lacasa, J., Martos, A., Tubau, F., Fernández, A., Viladrich, P. F. et al. (1995). Influence of dexamethasone on efficacy of ceftriaxone and vancomycin therapy in experimental pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 39, 2158–60.[Abstract]

12 . Paris, M., Hickey, S. M., Usher, M. I., Shelton, S., Olsen, K. D. & McCracken, G. H. (1994). Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 38, 1320–4.[Abstract]

13 . Mahajan, M., Rohatgi, D., Talwar, V., Patni, S. K., Mahajan, P. & Agarwal, D. S. (1997). Serum and cerebrospinal fluid concentrations of rifampicin at two dose levels in children with tuberculous meningitis. Journal of Communicable Diseases 29, 269–74.[Medline]

14 . Nau, R., Wellmer, A., Soto, A., Koch, K., Scheider, O., Schmidt, H. et al. (1999). Rifampicin reduces early mortality in experimental Streptococcus pneumoniae meningitis. Journal of Infectious Diseases 179, 1557–60.[ISI][Medline]

15 . Kucers, A., Crowe, S. M., Grayson, M. L. & Hoy, J. F. (1997). Rifampicin. In The Use of Antibiotics. A Clinical Review of Antibacterial, Antifungal and Antiviral Drugs, p. 690. Butterworth– Heinemann, Oxford.

16 . Ribera, E., Pou, L., Fernández-Solá, A., Campos, F., López, R. M., Ocaña, I. et al. (2001). Rifampicin reduces concentrations of trimethoprim and sulfamethoxazole in serum in human immunodeficiency virus-infected patients. Antimicrobial Agents and Chemotherapy 45, 3238–41.[Abstract/Free Full Text]

17 . Colmenero, J. D., Fernandez-Gallardo, L. C., Agundez, J. A., Sedeno, J., Benitez, J. & Valverde, E. (1994). Possible implications of doxycycline–rifampicin interaction for treatment of brucellosis. Antimicrobial Agents and Chemotherapy 38, 2798–802.[Abstract]

Received 6 July 2001; returned 26 October 2001; revised 11 December 2001; accepted 20 December 2001