a Departamento Micología, Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr Carlos G. Malbrán, Av. Velez Sarsfield 563, 1281 Buenos Aires; b Hospital J. A. Fernandez, Buenos Aires, Argentina
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Abstract |
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Introduction |
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Amphotericin B (AMB) and fluconazole (FCZ) are current acceptable therapies for cryptococcal meningitis. However, their effects remain suboptimal, and recurrence or treatment failure is still a problem. Recently, AMB plus flucytosine (5FC), for 2 weeks, followed by FCZ, was suggested as the treatment of choice.3 Another combination therapy proposed has been FCZ plus 5FC, which seemed to be clinically useful in patients with meningitis4 and in pulmonary cryptococcosis.5 On the other hand, a synergic interaction between AMB and rifampicin (RIF) has been demonstrated in vitro with other fungi such as Aspergillus6 and Candida spp.7
In a previous study, our group evaluated the in vitro activity of AMB against 16 isolates of C. neoformans obtained from AIDS patients with cryptococcal meningitis using timekill curves (TKCs), and by determining MICs and minimal fungicidal concentrations (MFCs). In that study,8 TKCs for AMB (1 mg/L) showed fungicidal activity against most of the isolates. Four isolates from patients who did not respond to conventional AMB therapy showed a persistent or tolerant effect. In spite of this, the MIC values obtained suggested that they were all susceptible.8
The aim of this study was to evaluate the interactive effects of combinations of drugs, namely, AMB plus 5FC, AMB plus RIF and FCZ plus 5FC, against five isolates of C. neoformans obtained from patients who died or failed to respond to AMB therapy.
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Materials and methods |
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The five isolates of C. neoformans included in this study were selected from separate AIDS patients with a first episode of cryptococcal meningitis who did not respond to AMB therapy and whose isolates appeared tolerant in TKCs obtained using AMB (Figure 1a). One patient died at the beginning of the AMB therapy, having received a total dosage of 50 mg (isolate 399). Three patients died during administration of AMB therapy, the total dosage received in all these cases being >500 mg. Isolates from these patients were assigned numbers 947, 1130 and 2672. In one patient, CSF culture still yielded C. neoformans (isolate 2294), despite the total dosage of AMB received being 750 mg.
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The following antifungal agents were used in the study: AMB (Squibb, New Brunswick, NJ, USA), 5FC (Sigma Chemical Co., St Louis, MO, USA), FCZ (Pfizer S.A., Buenos Aires, Argentina) and RIF (Hoechst Marion Roussel, Buenos Aires, Argentina). The drugs were provided as powders of known potency. Stock solutions were prepared as follows: AMB and FCZ were dissolved in 100% dimethylsulphoxide (DMSO; Sigma Chemical Co.) at concentrations of 1 g/L and 10 g/L, respectively; 5FC was dissolved in sterile distilled water at a concentration of 10 g/L; and RIF in 4:6 (v/v) methanolwater at a concentration of 5 g/L. Stock solutions were stored at 70°C until needed.
Timekill curves
Isolates were grown with shaking in RPMI 1640 (Sigma Chemical Co.) buffered with MOPS (Sigma Chemical Co.) to pH 7.0 for 18 h at 35°C. Initial inocula were adjusted to 1 McFarland scale (c. 106 cfu/mL). One mL of these inocula was diluted 10-fold in 9 mL of MOPS-buffered RPMI containing the drugs to be tested, alone and in combination, at the following final concentrations: AMB at 1 mg/L, FCZ at 10 mg/L, 5FC at 10 mg/L and RIF at 5 mg/L. A control growth tube (10 mL of RPMI, pH 7.0) without drugs was included in all experiments. The tubes were incubated at 35°C. Samples of 0.5 mL volume were removed from each of the tubes and subjected to serial 10-fold dilution at 0, 6, 12, 24, 48 and 72 h. From each of these serial 10-fold dilutions, 30 µL were plated on YM agar plates. After 72 h of incubation at 35°C colony counts were determined. A >99.9% reduction in the viable count compared with that seen at time zero was considered as the endpoint of the TKC. Tests were performed in duplicate.
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Results |
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The combination of AMB plus 5FC showed synergic activity against all the isolates, particularly 399 and 947. The other three isolates showed a decrease of 1 or 2 log at 24 h, which was maintained at 48 h with fungicidal activity at 72 h (Figure 1b).
The TKC for RIF alone was similar to the control growth curves (Figure 1c). When the combination of AMB plus RIF was evaluated, a very marked synergic effect was noted with four isolates, which were killed at 6 h. For one isolate (1130) a killing effect was detected at 12 h with regrowth being observed at 24 h (Figure 1c
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FCZ, at a concentration of 10 mg/L, failed to kill any of the isolates (Figure 2a). The combination of FCZ plus 5FC did not show any variation from the curve obtained with FCZ alone.
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Discussion |
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This in vitro study was designed to evaluate different available combinations of drugs against C. neoformans isolated from patients who failed initial AMB therapy. To analyse possible synergic effects of the combinations, TKCs were used. This approach allows the estimation of microbicidal activity, which in several studies with bacteria was found to be a more accurate determinant of clinical outcome than a simple numerical MIC or MBC.11
Recently, Van der Horst et al.,3 in a double-blind multicentre trial, determined that for initial treatment of AIDS-associated cryptococcal meningitis, the use of AMB plus 5FC was associated with an increased rate of CSF sterilization and decreased mortality at 2 weeks, as compared with AMB alone. Our in vitro results agree with this study, showing that the addition of a very low concentration of 5FC to AMB produced a synergic effect against tolerant isolates of C. neoformans. However, many patients cannot tolerate 5FC because of toxicity, primarily manifested as bone marrow suppression. Thus, alternative approaches would be highly desirable. Furthermore, because in the majority of cases therapy must be associated with the treatment of other infections, drug interactions occur and must be considered. In Argentina, tuberculosis is one of the most frequent infections in patients with AIDS. Therefore interaction between AMB and RIF (the first line agent for Mycobacterium tuberculosis) must be considered. Although RIF alone does not have antifungal activity, synergy with AMB has been previously demonstrated in vitro against Candida and Aspergillus spp.6 In this preliminary study, AMB plus RIF showed encouraging results; however, it will be useful to evaluate this synergic effect in vivo, by evaluating patients with tuberculosis plus cryptococcosis, who are receiving both drugs.
Recently, two clinical trials suggested the use of a combination of FCZ plus 5FC for pulmonary cryptococcosis and cryptococcal meningitis therapy.4,5 Our results did not show synergic interaction between these drugs against the isolates tested. However, it may be that synergy could not be detected at the low concentrations of drugs that were used. Further in vitro studies using higher concentrations for both drugs will be necessary to determine its usefulness.
Although more data are needed to evaluate the correlation between TKC and clinical outcome, these preliminary results suggest that for isolates tolerant to AMB, an alternative therapy could be considered.
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Acknowledgments |
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Notes |
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References |
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2 . Cahn, P., Cuatz, D., Guelfand, L., Kaufman, S. & Perez, H. (1995). Cryptococcal meningitis is a frequent first AIDS-defining illness with high rates of relapse in Argentina. In Program and Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 1990, Abstract I-98, p. 222. American Society for Microbiology, Washington, DC.
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Van der Horst, C., Saag, M. S., Cloud, G. A., Hamill, R. A., Graybill, J. R., Sobel, J. D. et al. (1997). Treatment of cryptococcal meningitis associated with immunodeficiency syndrome. New England Journal of Medicine 337, 1521.
4 . Mayanja-Kizza, H., Oishi, K., Mitarai, S., Yamashita, H., Nalongo, K., Watanabe, K. et al. (1998). Combination therapy with fluconazole and flucytosine for cryptococcal meningitis in Ugandan patients with AIDS. Clinical Infectious Diseases 26, 13626.[ISI][Medline]
5 . Bennett, J. E., Dismukes, W. E., Duma, R. J., Medoff, G., Sande, M. A., Gallis, H. et al. (1979). A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis. New England Journal of Medicine 301, 12631.[Abstract]
6 . Denning, D. W., Hanson, L. H., Perlman, A. M. & Stevens, D. A. (1992). In vitro susceptibility and synergy studies of Aspergillus species to conventional and new agents. Diagnosis in Microbiology and Infectious Diseases 15, 2134.
7 . Edwards, J. E., Jr, Morrison, J., Henderson, D. K. & Montgomerie, J. Z. (1980). Combined effect of amphotericin B and rifampin on Candida species. Antimicrobial Agents and Chemotherapy 17, 4847.[ISI][Medline]
8 . Rodero, L., Córdoba, S., Guelfand, L., Kaufman, S., Hochenfellner, F., Rossi, A. et al. (1996). In vitro susceptibility studies of Cryptococcus neoformans isolated from patients with AIDS. In Program and Abstracts of the Third International Conference of Cryptococcus and Cryptococcosis, Institut Pasteur, Paris, 1996. Abstract 3.2, p. 175. International Society for Human and Animal Mycology, The Netherlands.
9 . Powderly, W. G. (1992). Therapy for cryptococcal meningitis in patients with AIDS. Clinical Infectious Diseases 14, Suppl. 1, S549.[ISI][Medline]
10 . Nguyen, M. H., Barchiesi, F., McGough, D., Yu, V. L. & Rinaldi, M. G. (1995). In vitro evaluation of combination of fluconazole and flucytosine against Cryptococcus neoformans var. neoformans. Antimicrobial Agents and Chemotherapy 39, 16915.[Abstract]
11 . Johnson, C. C. (1996). In vitro testing: correlation between bactericidal susceptibility, body fluid levels and effectiveness of antibacterial therapy. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 81334. Williams & Wilkins, Baltimore, MD.
Received 30 March 1999; returned 19 July 1999; revised 27 September 1999; accepted 13 October 1999