In vitro susceptibility studies of Cryptococcus neoformans isolated from patients with no clinical response to amphotericin B therapy

L. Roderoa,*, S. Córdobaa, P. Cahnb, F. Hochenfellnera, G. Davela, C. Canterosa, S. Kaufmanb and L. Guelfandb

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activities of three antifungal drugs alone and in combination were evaluated against five isolates of Cryptococcus neoformans using time–kill curves (TKC). The isolates were from AIDS patients who had either died or had failed to show a clinical response during amphotericin B (AMB) treatment. AMB, fluconazole (FCZ) and flucytosine (5FC), and combinations of the drugs (AMB plus 5FC, AMB plus rifampicin (RIF) and FCZ plus 5FC), were evaluated. With all five isolates AMB did not show fungicidal activity; instead, a persistent or tolerant effect was observed. Combinations of AMB plus 5FC and AMB plus RIF showed a clear synergic effect, except for one isolate tested with AMB plus RIF. In contrast, the FCZ plus 5FC combination did not inhibit growth of any isolate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infections caused by Cryptococcus neoformans are an increasing problem in immunocompromised patients, particularly those with AIDS, in whom this organism is the fourth most common cause of life-threatening infection.1 Approximately 90% of AIDS patients infected with C. neoformans develop meningitis.1 In Buenos Aires city, Argentina, cryptococcal meningitis has been diagnosed in approximately 9% of patients with AIDS.2

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 time–kill 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Isolates

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 1aGo). 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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Figure 1. Time–kill curves of C. neoformans tested with amphotericin B (AMB) alone and in combination with flucytosine (5FC) and rifampicin (RIF): (a) for AMB; (b) for 5FC and the combination AMB plus 5FC; (c) for RIF and the combination AMB plus RIF. Control curves (dashed line), drug alone (dotted line) and combinations (continuous line) for isolates 947 ({triangleup}), 2672 ({diamond}), 399 ({circ}), 1130 ({square}) and 2294 (x).

 
Antifungal agents

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) methanol–water at a concentration of 5 g/L. Stock solutions were stored at –70°C until needed.

Time–kill 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.


    Results
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 Abstract
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 Materials and methods
 Results
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The TKC of the five isolates tested with AMB (1 mg/L) showed a low initial inhibition of growth. After 12 or 24 h of incubation, a reduction of <2 log was obtained. After this point, growth was resumed and at 48 or 72 h colony counts in the range 104–106 cfu/mL were detected (Figure 1aGo). None of the isolates was inhibited by 5FC (10 mg/L) (Figure 1bGo).

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 1bGo).

The TKC for RIF alone was similar to the control growth curves (Figure 1cGo). 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 1cGo).

FCZ, at a concentration of 10 mg/L, failed to kill any of the isolates (Figure 2aGo). The combination of FCZ plus 5FC did not show any variation from the curve obtained with FCZ alone.



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Figure 2. Time–kill curves of C. neoformans tested with fluconazole (FCZ) alone and in combination with flucytosine (5FC): (a) for FCZ; (b) for the combination FCZ plus 5FC. Control curves (dashed line), drug alone (dotted line) and combinations (continuous line) for isolates 947 ({triangleup}), 2672 ({diamond}), 399 ({circ}), 1130 ({square}) and 2294 (x).

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Although AMB and FCZ are useful therapies, the treatment of cryptococcal meningitis in AIDS patients is still a problem with high failure rates sometimes noted. There are a number of studies3,9 that have evaluated the possibility of more effective and less toxic alternatives. Combination therapy might be useful in these infections, involving immunocompromised hosts, in which enhanced drug activity is needed.10

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.


    Acknowledgments
 
We thank Marcelo Soria for helpful criticism. These results were presented at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, in September 1998 (Abstract 217 J).


    Notes
 
* Corresponding author. Tel/Fax: +54-11-4302-5066; E-mail: lrodero{at}mail.retina.ar

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    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Mitchell, T. G. & Perfect, J. R. (1995). Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8, 515–48.[Abstract]

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.

3 . 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, 15–21.[Abstract/Free Full Text]

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, 1362–6.[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, 126–31.[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, 21–34.

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, 484–7.[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, S54–9.[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, 1691–5.[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. 813–34. Williams & Wilkins, Baltimore, MD.

Received 30 March 1999; returned 19 July 1999; revised 27 September 1999; accepted 13 October 1999