Development of Secondary Resistance to Fluconazole in Cryptococcus neoformans Isolated from a Patient with AIDS

ALVES, Sydney H.; LOPES, Jorge O.; COSTA, Jane M.; KLOCK, Clóvis

doi:10.1590/S0036-46651997000600010

Abstracts

Cryptococcus neoformans is the fifth most common opportunistic agent of infection in patients with AIDS in the USA, exceeded only by Candida species, Pneumocystis carinii, cytomegalovirus and Mycobacterium avium1, 2, 6, 10, 11. In Brazil is the sixth, exceeded by Candida species, P. carinii, Mycobacterium species, Toxoplasma gondii, and herpes simplex virus (AIDS, Boletim Epidemiológico, set/nov 96, Ministério da Saúde, Brasil). During 30 years, the treatment of C. neoformans meningitis was based on the use of amphotericin B with or without flucytosine13. Nowadays, with the immunodepression caused by human immunodeficiency virus (HIV) infection and the availability of new antifungal drugs as the triazoles, the concept related to cure and relapses of cryptococcosis has been altered7, 20. Patients are treated with amphotericin B with or without flucytosine as initial therapy, but maintenance therapy is always necessary in AIDS patients with C. neoformans infections

Cryptococcus neoformans; Secondary resistance; Fluconazole

Relatamos um caso de meningite por Cryptococcus neoformans em paciente com Síndrome de Imunodeficiência Adquirida (SIDA). A terapia de manutenção com fluconazol não evidenciou melhora clínica e micológica, ao mesmo tempo em que o teste de suscetibilidade in vitro revelou aumento progressivo da concentração inibitória mínima (CIM). Estes resultados sugerem o desenvolvimento de resistência secundária ao fluconazol, todavia, resistência cruzada com outros derivados azólicos não foi constatada

BRIEF COMMUNICATION

DEVELOPMENT OF SECONDARY RESISTANCE TO FLUCONAZOLE IN Cryptococcus neoformans ISOLATED FROM A PATIENT WITH AIDS

Sydney H. ALVES, Jorge O. LOPES, Jane M. COSTA & Clóvis KLOCK

KEYWORDS: Cryptococcus neoformans; Secondary resistance; Fluconazole.

Cryptococcus neoformans is the fifth most common opportunistic agent of infection in patients with AIDS in the USA, exceeded only by Candida species, Pneumocystis carinii, cytomegalovirus and Mycobacterium avium^{1, 2, 6, 10, 11}. In Brazil is the sixth, exceeded by Candida species, P. carinii, Mycobacterium species, Toxoplasma gondii, and herpes simplex virus (AIDS, Boletim Epidemiológico, set/nov 96, Ministério da Saúde, Brasil). During 30 years, the treatment of C. neoformans meningitis was based on the use of amphotericin B with or without flucytosine¹³. Nowadays, with the immunodepression caused by human immunodeficiency virus (HIV) infection and the availability of new antifungal drugs as the triazoles, the concept related to cure and relapses of cryptococcosis has been altered^{7, 20}. Patients are treated with amphotericin B with or without flucytosine as initial therapy, but maintenance therapy is always necessary in AIDS patients with C. neoformans infections.

We communicate a case of C. neoformans meningitis in a patient with AIDS whose successive isolations during maintenance therapy with fluconazole showed reduction on susceptibility to the drug.

A 30 year-old white man with HIV infection since 1987, related to intravenous drug abuse, was hospitalized at the Santa Maria University Hospital on January 13, 1995. He has suffered from tuberculosis since 1994, and on that occasion complained of headache, nausea, projectile vomiting and fever (39°C). Cerebrospinal fluid (CSF) examination revealed C. neoformans, isolated on Sabouraud dextrose agar and characterized as var. neoformans on canavanine-glicine medium. The patient was treated with intravenous amphotericin B 50 mg/day during six weeks, and later with oral fluconazole 200 mg/day as maintenance therapy.

On May 19, 1995, he was hospitalized again with similar clinical history, and C. neoformans was isolated from CSF. Broth dilution susceptibility testing¹⁶ revealed a MIC of 0.5 µg/ml for fluconazole, itraconazole, ketoconazole and amphotericin B, and the dosage was maintained without improvement of clinical symptoms. On May 30, CSF was collected again and C. neoformans isolation revealed a MIC of 1.0 µg/ml. Even with dosage of 400 mg/day, C. neoformans was isolated again on the 17^th day. A MIC of 32 µg/ml for fluconazole and 0.5 µg/ml for other azoles and amphotericin B was detected, and the drug was then substituted by amphotericin B.

C. neoformans isolate was tested for in vitro susceptibility by standard method proposed by NCCLS (proposed standard M27-P)¹⁶. Antifungal agents were provided by manufacturers as pure powder form. Isolates were tested by a broth macrodilution technique with RPMI 1640 medium containing L-glutamine but no sodium bicarbonate and buffered to pH 7.0 with 0.165M morpholinopropane-sulphonic acid (MOPS). An inoculum of 10³ cells/ml was prepared by a spectrophotometric method, and incubation was at 35°C for 72h. Final drug concentrations ranged from 64 to 0.125 µg/ml. The MICs were determined after incubation: for amphotericin B the MIC was the lowest concentration that inhibited the growth. Azoles MICs were defined as the lowest drug concentrations which resulted in a visual turbidity less than or equal to 80% inhibition compared with that produced by growth control which was obtained by diluting at 1:5 the drug-free control growth, with RPMI broth.

The treatment of C. neoformans meningitis remains a frustrating problem, considering limitations and effectiveness on therapy. In AIDS patients cure has not been achieved, and studies indicated that cryptococcosis is highly refractory to therapy and associated with a relapse rate of more than 50% after a primary treatment^{7, 22}. The treatment includes an initial therapy with amphotericin B, followed by a maintenance therapy with an azole derivative, such as fluconazole or itraconazole^{7, 20}. Therapy with amphotericin B or fluconazole alone is inefficient¹⁴, but the association has been an alternative therapeutic option⁷. In addition, the possibility of resistance of C. neoformans to antifungal drugs make the management of these patients more problematic. C. neoformans resistance to amphotericin B related to alterations on the quality of cytoplasmic membrane sterol has been reported^{3, 11, 12, 21}.

Primary resistance of yeast to azoles derivatives, specially fluconazole, is an emerging phenomenon, well described in Candida krusei and C. glabrata²³ and may represent the tip of an iceberg¹⁷. Alterations on susceptibility of C. neoformans to fluconazole have been described^{6, 8, 18, 20} and have been attributed mainly to efflux phenomenon in which drug loss from cellular interior occurs by active transport and is mediated by glicoprotein P or by other proteins such as MFS (major facilitator superfamily)⁵. Another resistance mechanism to azole agents include: a) reduced permeability of the membrane resulting from changes in membrane sterol composition with consequent minor capture of the drug by the fungus; b) a mutation in the target fungal enzyme (sterol 14-a demethylase, a cytochrome P450 enzyme) resulting in decreased binding affinity for the azole drugs; c) an overproduction of the target cytochrome P450 enzyme; d) amplification of CYP 51 gene; e) D^{5, 6} desaturase alterations and D^{8, 7} isomerase lesions, among others^{17, 23}.

In this report we must emphasize that neither azoles cross resistance was observed nor resistance among amphotericin B and azoles. In view of these facts we may suppose that ergosterol biosynthesis pathway remained unaltered, reinforcing the idea that efflux can be the main mechanism of fluconazole-resistance.

In Brazil, FRANZOT & HAMDAN⁹ studied the susceptibility of 53 strains of C. neoformans from clinical and environmental isolates and showed that the fluconazole MIC range for clinical strains were 0.5-16 µg/ml which in our opinion encompass resistant strains. So, the present fluconazole-resistant strain, the elevated MICs showed by those authors⁹ and the frequent fluconazole failures on cryptococcosis meningitis in AIDS patients^{1, 6, 7, 8, 14, 15} support the idea that even in Brazil C. neoformans fluconazole-resistant strains may be more frequent than has been supposed.

Chemotherapy alone is not adequate for all patients with cryptococcosis⁷. In the present report, relapse of cryptococcal meningitis reflects deterioration on immune defenses, incapable of eradicate the infection^{4, 15}, since C. neoformans strains remained susceptible to amphotericin B. Alternative treatment regimens have been developed or are under investigation, including induction-consolidation regimen using several drugs^{4, 7}. On the other hand, additional studies are necessary on prophylactic therapy with the purpose to detect interference on initial susceptibility of C. neoformans or other fungi²⁰.

RESUMO

Desenvolvimento de resistência secundária ao fluconazol em Cryptococcus neoformans isolado de paciente com AIDS

Relatamos um caso de meningite por Cryptococcus neoformans em paciente com Síndrome de Imunodeficiência Adquirida (SIDA). A terapia de manutenção com fluconazol não evidenciou melhora clínica e micológica, ao mesmo tempo em que o teste de suscetibilidade in vitro revelou aumento progressivo da concentração inibitória mínima (CIM). Estes resultados sugerem o desenvolvimento de resistência secundária ao fluconazol, todavia, resistência cruzada com outros derivados azólicos não foi constatada.

Recebido para publicação em 14/02/1997

Aceito para publicação em 17/12/1997

Serviço de Micologia, Hospital Universitário, Universidade Federal de Santa Maria, Rio Grande do Sul, Brasil.

Correspondence to: Jorge O. Lopes. Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, 97119-970 Santa Maria, RS, Brasil.

1. BAYLLY, M. P.; BOIBIEUX, A.; BIRON, F. et al. Persistence of Cryptococcus neoformans in the prostate: failure of fluconazole despite high doses. J. infect. Dis., 164: 435-436, 1991.
2. BERRY, A. J.; RINALDI, M. G. & GRAYBILL, J. R. Use of high-dose fluconazole as salvage therapy for cryptococcal meningitis in patients with AIDS. Antimicrob. Agents Chemother., 36: 690-692, 1992.
3. BODENHOFF, J. Development of strains of Cryptococcus neoformans resistant to nystatin, amphotericin B, trichomycin and polymixin B. Acta path. microb. scand., 73: 572-582, 1968.
4. CASADEVALL, A.; SPITZER, E. D.; WEBB, D. & RINALDI, M. G. Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrob. Agents Chemother., 37: 1383-1386, 1993.
5. CLARCK, F. S.; PARKINSON, T.; HITCHCOCK, C. A. & GOW, N. A. R. Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance. Antimicrob. Agents Chemother., 40: 419-425, 1996.
6. COKER, R. J. & HARRIS, J. R. W. Failure of fluconazole treatment in cryptococcal miningitis despite adequate CSF levels. J. Infect., 23: 101-102, 1991.
7. DISMUKES, W. E. Management of cryptococcosis. Clin. infect. Dis., 17 (suppl. 2): S507-S512, 1993.
8. DUPONT, B.; DENNING, D. W.; MARRIOTT, D. et al. Mycosis in AIDS patients. J. med. vet. Mycol., 32 (suppl. 1): 65-77, 1994.
9. FRANZOT, S. P. & HAMDAN, J. S. In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs. Antimicrob. Agents Chemother., 40: 822-824, 1996.
10. HAUBRICH, R. H.; HAGHIGHAT, D.; BOZZETTE, S. A. et al. High-dose fluconazole for treatment of cryptococcal disease in patients with Human Immunodeficiency Virus infection. J. infect. Dis., 170: 238-242, 1994.
11. JOSEPH-HORNE, T.; HOLLOMON, D.; LOEFFLER, R. S. T. & KELLY, S. L. Cross-resistance to polyene and azole drugs in Cryptococcus neoformans Antimicrob. Agents Chemother., 39: 1526-1529, 1995.
12. JOSEPH-HORNE, T.; LOEFFLER, R. S. T.; HOLLOMON, D. W. & KELLY, S. L. Amphotericin B resistant isolates of Cryptococcus neoformans without alteration in sterol biosynthesis. J. med. vet. Mycol., 34: 223-225, 1996.
13. KWON-CHUNG, K. J.; PFEIFFER, T.; CHANG, Y. C. et al. Molecular biology of Cryptococcus neoformans and therapy of cryptococcosis. J. med. vet. Mycol., 32 (suppl. 1): 407-415, 1994.
14. LARSEN, R. A.; BOZZETTE, S. A. & JONES, B. E. Fluconazole combined with flucytosine for treatment of cryptococcal meningitis in patients with AIDS. Clin. infect. Dis., 19: 741-745, 1994.
15. MITCHELL, T. G. & PERFECT, J. R. Cryptococcosis in the era of AIDS 100 years after discovery of Cryptococcus neoformans Clin. Microbiol. Rev., 8: 515-548, 1995.
16. NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS Reference method for broth dilution antifungal susceptibility testing of yeasts: proposed standard Vilanova, 1992. 22p. (NCCLS Document M27-P).
17. ODDS, F. C. Resistance of yeasts to azole-derivative antifungals. J. Antimicrob. Chemother., 31: 463-471, 1993.
18. PAUGAM, A.; DUPOUY-CAMET, J.; BLANCHE, P. et al. Increased fluconazole resistance of Cryptococcus neoformans isolated from a patient with AIDS and recurrent meningitis. Clin. infect. Dis., 19: 976-977, 1994.
19. PEETERMANS, W.; BOBBAERS, H.; VERHAEGEN, J. & VANDEPITTE, J. Fluconazole-resistant Cryptococcus neoformans var. gatti in an AIDS patient. Acta clin. belg., 48: 405-409, 1993.
20. PINNER, R. W.; HAJJEH, R. A. & POWDERLY, W. G. Prospects of prevening cryptococcosis in persons infected with Human Immunodeficiency Virus. Clin. infect. Dis., 21 (suppl. 1): S103-S107, 1995.
21. POWDERLY, W. G.; KEATH, E. J.; SOKOL-ANDERSON, M. et al. Amphotericin B resistant Cryptococcus neoformans in a patient with AIDS. Infect. Dis. clin. Pract., 1: 314-316, 1990.
22. STERN, J. J.; HARTMAN, B. J.; SHARKEY, P. et al Oral fluconazole therapy for patients with acquired immunodeficiency syndrome and cryptococcosis: experience with 22 patients. Amer. J. Med., 85: 477-480, 1988.
23. VANDEN BOSSCHE, H.; WARNOCK, D. W.; DUPONT, B. et al. Mechanisms and clinical impact of antifungal drug resistance. J. med. vet. Mycol., 32 (suppl. 1): 189-202, 1994.

Publication Dates

Publication in this collection
09 Oct 1998
Date of issue
Nov 1997

History

Received
14 Feb 1997
Accepted
17 Dec 1997

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BAYLLY, M. P.; BOIBIEUX, A.; BIRON, F. et al. Persistence of Cryptococcus neoformans in the prostate: failure of fluconazole despite high doses. J. infect. Dis., 164: 435-436, 1991.

[2] 2. BERRY, A. J.; RINALDI, M. G. & GRAYBILL, J. R. Use of high-dose fluconazole as salvage therapy for cryptococcal meningitis in patients with AIDS. Antimicrob. Agents Chemother., 36: 690-692, 1992.

[3] 3. BODENHOFF, J. Development of strains of Cryptococcus neoformans resistant to nystatin, amphotericin B, trichomycin and polymixin B. Acta path. microb. scand., 73: 572-582, 1968.

[4] 4. CASADEVALL, A.; SPITZER, E. D.; WEBB, D. & RINALDI, M. G. Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrob. Agents Chemother., 37: 1383-1386, 1993.

[5] 5. CLARCK, F. S.; PARKINSON, T.; HITCHCOCK, C. A. & GOW, N. A. R. Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance. Antimicrob. Agents Chemother., 40: 419-425, 1996.

[6] 6. COKER, R. J. & HARRIS, J. R. W. Failure of fluconazole treatment in cryptococcal miningitis despite adequate CSF levels. J. Infect., 23: 101-102, 1991.

[7] 7. DISMUKES, W. E. Management of cryptococcosis. Clin. infect. Dis., 17 (suppl. 2): S507-S512, 1993.

[8] 8. DUPONT, B.; DENNING, D. W.; MARRIOTT, D. et al. Mycosis in AIDS patients. J. med. vet. Mycol., 32 (suppl. 1): 65-77, 1994.

[9] 9. FRANZOT, S. P. & HAMDAN, J. S. In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs. Antimicrob. Agents Chemother., 40: 822-824, 1996.

[10] 10. HAUBRICH, R. H.; HAGHIGHAT, D.; BOZZETTE, S. A. et al. High-dose fluconazole for treatment of cryptococcal disease in patients with Human Immunodeficiency Virus infection. J. infect. Dis., 170: 238-242, 1994.

[11] 11. JOSEPH-HORNE, T.; HOLLOMON, D.; LOEFFLER, R. S. T. & KELLY, S. L. Cross-resistance to polyene and azole drugs in Cryptococcus neoformans Antimicrob. Agents Chemother., 39: 1526-1529, 1995.

[12] 12. JOSEPH-HORNE, T.; LOEFFLER, R. S. T.; HOLLOMON, D. W. & KELLY, S. L. Amphotericin B resistant isolates of Cryptococcus neoformans without alteration in sterol biosynthesis. J. med. vet. Mycol., 34: 223-225, 1996.

[13] 13. KWON-CHUNG, K. J.; PFEIFFER, T.; CHANG, Y. C. et al. Molecular biology of Cryptococcus neoformans and therapy of cryptococcosis. J. med. vet. Mycol., 32 (suppl. 1): 407-415, 1994.

[14] 14. LARSEN, R. A.; BOZZETTE, S. A. & JONES, B. E. Fluconazole combined with flucytosine for treatment of cryptococcal meningitis in patients with AIDS. Clin. infect. Dis., 19: 741-745, 1994.

[15] 15. MITCHELL, T. G. & PERFECT, J. R. Cryptococcosis in the era of AIDS 100 years after discovery of Cryptococcus neoformans Clin. Microbiol. Rev., 8: 515-548, 1995.

[16] 16. NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS Reference method for broth dilution antifungal susceptibility testing of yeasts: proposed standard Vilanova, 1992. 22p. (NCCLS Document M27-P).

[17] 17. ODDS, F. C. Resistance of yeasts to azole-derivative antifungals. J. Antimicrob. Chemother., 31: 463-471, 1993.

[18] 18. PAUGAM, A.; DUPOUY-CAMET, J.; BLANCHE, P. et al. Increased fluconazole resistance of Cryptococcus neoformans isolated from a patient with AIDS and recurrent meningitis. Clin. infect. Dis., 19: 976-977, 1994.

[19] 19. PEETERMANS, W.; BOBBAERS, H.; VERHAEGEN, J. & VANDEPITTE, J. Fluconazole-resistant Cryptococcus neoformans var. gatti in an AIDS patient. Acta clin. belg., 48: 405-409, 1993.

[20] 20. PINNER, R. W.; HAJJEH, R. A. & POWDERLY, W. G. Prospects of prevening cryptococcosis in persons infected with Human Immunodeficiency Virus. Clin. infect. Dis., 21 (suppl. 1): S103-S107, 1995.

[21] 21. POWDERLY, W. G.; KEATH, E. J.; SOKOL-ANDERSON, M. et al. Amphotericin B resistant Cryptococcus neoformans in a patient with AIDS. Infect. Dis. clin. Pract., 1: 314-316, 1990.

[22] 22. STERN, J. J.; HARTMAN, B. J.; SHARKEY, P. et al Oral fluconazole therapy for patients with acquired immunodeficiency syndrome and cryptococcosis: experience with 22 patients. Amer. J. Med., 85: 477-480, 1988.

[23] 23. VANDEN BOSSCHE, H.; WARNOCK, D. W.; DUPONT, B. et al. Mechanisms and clinical impact of antifungal drug resistance. J. med. vet. Mycol., 32 (suppl. 1): 189-202, 1994.