The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.Comparou-se a suscetibilidade de 69 amostras de C. neoformans isoladas de pacientes com SIDA com 13 amostras de C. neoformans isoladas do meio ambiente, frente a quatro agentes antifúngicos. Com base na metodologia preconizada pelo NCCLS (M27-A) as concentrações inibitórias mínimas (CIMs) obtidas para a anfotericina B, itraconazol e cetoconazol foram muito semelhantes nos dois grupos estudados. Todavia, frente ao fluconazol, os isolados clínicos evidenciaram menor sensibilidade do que os provenientes do meio ambiente. Alguns aspectos envolvendo a importância e dificuldades dos testes de suscetibilidade com Cryptococcus neoformans são também discutidos pelos autores

ALVES, Sydney Hartz

CASALI, Agnes Kiesling

COSTA, Jane M.

LUBECK, Irina

OLIVEIRA, Loiva T.

VAINSTEIN, Marilene Henning

English

Cadernos Espinosanos (E-Journal)

		$$"	 !	""#(1) Prof. Adjunto Microbiologia. Departamento Análises Clínicas e Toxicológicas; Laboratório de Pesquisas Micológicas (LAPEMI). Universidade Federal de Santa Maria (UFSM), SantaMaria, RS, Brasil.(2) Hospital Universitário de Santa Maria, Santa Maria, RS, Brasil.(3) Acadêmica do Curso de Veterinária, Bolsista do PBIC (CNPq), UFSM, Santa Maria, RS, Brasil.(4) Programa de Pós-graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil.Correspondence to: Sydney Hartz Alves, Departamento de Análises Clínicas e Toxicológicas, CCS, Universidade Federal de Santa Maria. 97119-000 Santa Maria, RS, Brazil. E-mail:hartzsa@fatecnet.ufsm.brIn vitro SUSCEPTIBILITY TO ANTIFUNGAL AGENTS OF CLINICAL AND ENVIRONMENTALCryptococcus neoformans ISOLATED IN SOUTHERN OF BRAZILSydney Hartz ALVES(1), Loiva T. OLIVEIRA(2), Jane M. COSTA(2), Irina LUBECK(3), Agnes Kiesling CASALI(4) & Marilene Henning VAINSTEIN(4)SUMMARYThe purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformansstrains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole werevery similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates.The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testingare also discussed.KEYWORDS: Cryptococcus neoformans; Susceptibility; Antifungal agentsINTRODUCTIONCryptococcus neoformans is an opportunistic yeast recognized asthe etiological agent of human cryptococcosis. This widespread organismwas noted to occur in immunosuppressed hosts especially those withAIDS, for whom disseminated disease is being increasingly reported25.Cryptococcus neoformans is rarely isolated from healthy individualsand does not appear to be a common human commensal. The sporadicnature of human cryptococcosis, the extreme rarity of documentedhuman-to-human transmission events3,12 and the high prevalence of C.neoformans in the environment indicate that human infection is acquiredfrom environmental sources5. It was assumed that inhalation of infectiousparticles from avian excreta, the main natural source of environmentalstrains of the var. neoformans, is the major route for human infection5 .Based on the strong relationship between environmental and clinicalC. neoformans strains5 and the few comparative studies carried out onantifungal susceptibilities9,10, the purpose of this study was to evaluatethe susceptibility of 82 C. neoformans strains isolated in the state of RioGrande do Sul, Brazil.MATERIAL AND METHODSCryptococcus neoformans strains: a total of 82 strains were studied.Forty-eight isolates were obtained from cerebrospinal fluid (43), blood(3) and sputum (2) from 38 AIDS patients at the Hospital Universitáriode Santa Maria, Santa Maria (RS, Brazil) from January 1996 throughDecember 2000. Thirty-eight strains were obtained from patients duringthe initial diagnosis of cryptococcal infection and ten strains wereobtained after the patients had been treated with amphotericin B orfluconazole. Other twenty-one clinical samples were kindly providedby Central Laboratory of Instituto de Pesquisas Biológicas da Secretariada Saúde e Meio Ambiente in Rio Grande do Sul State – LACEN(fourteen) and by Hospital São Lucas (seven), from Porto Alegre.Environmental samples (thirteen) were from pigeon excreta originatedfrom two different cities in the Rio Grande do Sul State, Porto Alegreand Santa Cruz do Sul, located in the South region of Brazil and separatedapart by a distance of 150 km. For C. neoformans isolation, 1.0 g ofweathered pigeon excreta was added to 10 ml sterilized saline solutionwith both chloramphenicol and ampicillin at 150 mg ml–1 each. Afterfiltration through sterilized gauze, aliquots of 10–1 and 10–2 dilutionswere inoculated onto birdseed agar plates and incubated at 37 °C up to21 days. All strains were identified as C. neoformans by a positive Nigerseed agar response, positive urease test, ability to grow at 37 °C andnegative nitrogen test. The profiles of carbon compound assimilationwere also determined5. Each isolate was identified as C. neoformans varneoformans by the canavanine-glycine-bromothymol-blue-agar methoddescribed by KWON-CHUNG et al.18.Antifungal agents: the antifungal agents used were amphotericin B(Sigma), itraconazole and ketoconazole (Jansen Pharmaceutica), andfluconazole (Pfizer).Susceptibility testing: we used the macrodilution technique19. RPMI1640 medium (American Biorganics Inc.) containing L-glutamine wasprepared according to manufacturer instructions. After reconstitutionthe medium was supplemented with glucose to obtain a final8@ALVES, S.H.; OLIVEIRA, L.T.; COSTA, J.M.; LUBECK, I.; CASALI, A.K. & VAINSTEIN, M.H. - In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcusneoformans isolated in Southern of Brazil. Rev. Inst. Med. trop. S. Paulo, 43(5):267-270, 2001.concentration of 2%, and buffered to pH 7.0 with 3-(N-morpholino)propanesulfonic acid (MOPS; Sigma) to a final concentrationof 165 mM. The procedures of inoculum preparation and incubationwere those of the M27-A methodology19. The minimal inhibitoryconcentration (MIC) of amphotericin B was defined as the lowestconcentration of drug which resulted in complete inhibition of visiblegrowth. The MICs of azoles were defined as the lowest concentration ofdrug which resulted in an 80% reduction of fungal growth compared tocontrol. The assays were read 48 h after inoculation. The data are reportedas MIC ranges and MICs at which 50% and 90% of the isolates wereinhibited. Candida parapsilosis ATCC 22019 and Candida krusei ATCC6258 were used as quality control for the susceptibility tests.RESULTSTable 1 summarizes the in vitro susceptibilities of 69 clinical and 13environmental isolates.Amphotericin B showed similar MIC ranges against the strains fromboth sources but MIC 50% and MIC 90% were slightly more elevatedfor the environmental strains.Fluconazole showed a broad MIC range, varying from 0.125 to16 g/ml for clinical and environmental isolates. The MICs 50% and90% were more elevated for clinical than environmental isolates.For itraconazole the MICs were within narrow ranges (0.031-0.25 g/ml) for clinical and environmental isolates and the MIC 50%was the same for all strains. The MIC 90% for clinical isolates was moreelevated than environmental isolates. Ketoconazole showed narrow MICranges from 0.031 g/ml to 0.5 g/ml. The MIC 50% for clinical isolateswas more elevated than environmental isolates, but the MIC 90% wassimilar for clinical and environmental strains.DISCUSSIONThe concern about C. neoformans susceptibility is not recent4;however considering the newly standardized testing, the results currentlyobtained are becoming more appreciated because they allow inter-laboratory comparisons. Our purpose was to compare clinical andenvironmental isolates because the emergence of resistance is anapparently rare phenomenon that encompasses development after longtreatment with antifungals (secondary resistance) or primary resistancewithout former exposure to these agents8,23.Our results are closely similar to those reported by otherauthors7,9,12,14,27 and seem to indicate susceptibility of C. neoformansstrains to antifungal agents. However, some aspects about susceptibilitytests deserve attention.The narrow MIC ranges of amphotericin B have been pointed out tobe a consequence of RPMI 1640 medium which may not be a goodculture medium to warrant good C. neoformans growth and thus, couldbe hindering the detection of resistance8,11,23 . Reports about amphotericinB-resistant C. neoformans are scarce15,16,17,24 even during the AIDS era.Based on the M27-A methodology, our MIC ranges obtained foramphotericin B allow us to conclude that the clinical and environmentalisolates studied are susceptible to the drug, as also reported by others7,9,14.Fluconazole has been shown to be an effective alternative toamphotericin B in the treatment of cryptococcal meningitis and is themost commonly used antifungal agent in maintenance therapy for thisdisease25. The majority of cases of meningitis due to C. neoformansresistant to fluconazole have been reported in AIDS patients after longtreatments or prophylaxis with fluconazole1,2,21,22. Furthermore, afluconazole resistant strain isolated from an immunocompetent patientwithout exposure to this triazole has been reported, which alerts thatenvironmental strains can be primarily resistant to fluconazole20. So,cryptococcal susceptibility to fluconazole could be an important predictorof treatment success and MICs can be useful to monitor the possibledevelopment of resistance during therapy and to identify primaryresistance8,28. Our results based on MIC 50% and MIC 90% show thatclinical isolates were less susceptible to fluconazole than environmentalisolates even though MIC  16 g/ml was observed in both groups ofstrains. However, the overall susceptibility of the southern Brazilian C.neoformans isolates to fluconazole was very similar to that reported forAmerican isolates and in another Brazilian study9,27,28. FRANZOT &HAMDAN9 found that 100% of isolates from Minas Gerais (a centralBrazilian state) were inhibited by  16 g/ml of fluconazole, as alsoobserved in the present investigation.C. neoformans is extremely susceptible to itraconazole in vitro26;our data are similar to those reported by several other investigators whoused the same methodology7,9. It seems clear that C. neoformansresistance to itraconazole is too rare and cross-resistance with fluconazoledoes not occur1,6,21,22. However, IWATA et al.13 obtained an itraconazole–resistant C. neoformans strain after exposure to N-nitro-nitrosoguanidine.The mechanism of action of itraconazole which blocks the lanosterol14 -demethylase and the NADPH-dependent-3-ketosteroid reductasein C. neoformans may have implications both for its potent antifungalactivity and for its reduced development of cross-resistance comparedto other azoles5. We did not observe differences in itraconazole MICsTable 1In vitro susceptibility of C. neoformans strains to amphotericin B and azolesAntifungal agents and MIC (µg/ml)astrains groupb Range 50% 90%Amphotericin BClinical 0.0625-0.5 0.125 0.25Environmental 0.0625-0.5 0.25 0.5FluconazoleClinical 0.125- 16 2.0 8.0Environmental 0.125- 16 0.5 4.0ItraconazoleClinical 0.031-0.25 0.125 0.25Environmental 0.031-0.25 0.125 0.125KetoconazoleClinical 0.031-0.5 0.125 0.125Environmental 0.031-0.25 0.0625 0.125a 50% and 90%, MICs at which 50 and 90% of the strains, were inhibited,respectively; b Clinical isolates (n = 69); environmental isolates (n = 13).ALVES, S.H.; OLIVEIRA, L.T.; COSTA, J.M.; LUBECK, I.; CASALI, A.K. & VAINSTEIN, M.H. - In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcusneoformans isolated in Southern of Brazil. Rev. Inst. Med. trop. S. Paulo, 43(5):267-270, 2001.850% between clinical and environmental isolates and our results aresimilar to those reported by others5,9.Ketoconazole is not indicated for the treatment of cryptococcalmeningoencephalitis but was included in the present study in order toevaluate a possible cross-resistance among azoles. The ketoconazoleMICs observed were as low as that obtained for itraconazole and theinterpretation is the same. The 3 C. neoformans strains that showedfluconazole MICs = 16 g/ml did not demonstrate elevated MICs foritraconazole and ketoconazole.Finally, we found that clinical and environmental C. neoformansstrains showed a similar pattern of susceptibility to amphotericin B,itraconazole and ketoconazole. The clinical isolates were less susceptibleto fluconazole than the environmental isolates, possibly as a consequenceof therapy with this agent. As postulated by others, during therapy ofcryptococcosis the susceptibility tests of recurrent isolates may monitorthe development of secundary resistance, because if susceptibility doesnot predict successful therapy, resistance should often predict therapeuticfailure1,2,6,8,15,16,19,21,22,23,24,28.RESUMOSuscetibilidade in vitro a antifúngicos, de amostras clínicas eambientais de Cryptococcus neoformans isoladas no sul do BrasilComparou-se a suscetibilidade de 69 amostras de C. neoformansisoladas de pacientes com SIDA com 13 amostras de C. neoformansisoladas do meio ambiente, frente a quatro agentes antifúngicos. Combase na metodologia preconizada pelo NCCLS (M27-A) as concentraçõesinibitórias mínimas (CIMs) obtidas para a anfotericina B, itraconazol ecetoconazol foram muito semelhantes nos dois grupos estudados.Todavia, frente ao fluconazol, os isolados clínicos evidenciaram menorsensibilidade do que os provenientes do meio ambiente. Alguns aspectosenvolvendo a importância e dificuldades dos testes de suscetibilidadecom Cryptococcus neoformans são também discutidos pelos autores.REREFENCES1. ARMENGOU, A.; POCAR, C.; MASCARÓ, J. & GARCIA-BRAGADO, F. – Possibledevelopment of resistance to fluconazole during suppressive therapy for AIDS-associated cryptococcal meningitis. Clin. infect. Dis., 23: 1337-1338, 1996.2. BERG, J.; CLANCY, C.J. & NGUYEN, M.H. – The hidden danger of primary fluconazoleprophylaxis for patients with AIDS. Clin. infect. Dis., 26: 186-187, 1998.3. BEYT Jr., B.E. & WALTMAN, S.R. – Cryptococcal endophthalmitis after cornealtransplantation. New Engl. J. Med., 298: 825-826, 1978.4. BODENHOFF, J. – Development of strains of Cryptococcus neoformans resistant tonystatin, amphotericin B, trichomycin and polymyxin B. Acta path. microbiol.scand., 73: 572-582, 1968.5. CASADEVALL, A. & PERFECT, J.R. – Cryptococcus neoformans. Washington,American Society of Microbiology, 1998.6. COKER, R.J. & HARRIS, J.R.W. - Failure of fluconazole treatment in cryptococcalmeningitis despite adequate CSF levels. J. Infect., 23: 101-103, 1991.7. DAVEY, K.G.; HOLMES, A.D.; JOHNSON, E.M.; SZEKELY, A. & WARNOCK, D.W.– Comparative evaluation of Fungitest and broth microdilution methods for antifungaldrug susceptibility testing of Candida species and Cryptococcus neoformans. J. clin.Microbiol., 36: 926-930, 1998.8. ESPINEL-INGROFF, A. – Clinical relevance of antifungal resistance. Infect. Dis. Clin.N. Amer., 11: 929-944, 1997.9. FRANZOT, S.P. & HAMDAN, J.S. – In vitro susceptibilities of clinical and environmentalisolates of Cryptococcus neoformans to five antifungal drugs. Antimicrob. AgentsChemother., 40: 822-824, 1996.10. FROMTLING, R.A.; ABRUZZO, G.K. & RUIZ, A. – Virulence and antifungalsusceptibility of environmental and clinical isolates of Cryptococcus neoformansfrom Puerto Rico. Mycopathologia (Den Haag), 106: 163-166, 1989.11. GHANNOUM, M.A.; IBRAHIM, A.S.; FU, Y.; SCHAFIQ, M. et al. – Susceptibilitytesting of Cryptococcus neoformans: a microdilution technique. J. clin. Microbiol.,30: 2881-2886, 1992.12. GLASER, J.B. & GARDEN, A. – Inoculation of cryptococcosis without transmission ofthe acquired immunodeficiency syndrome. New Engl. J. Med., 313: 266, 1985.13. IWATA, K.; YAMASHITA, T.; OHSUMI, M.; BABA, M. et al. – Comparativemorphological and biological studies on the itraconazole- and ketoconazole-resistantmutants of Cryptococcus neoformans. J. med. vet. Mycol., 28: 77-90, 1990.14. JESSUP, C.J.; PFALLER, M.A.; MESSER, S.A. et al. – Fluconazole susceptibility testingof Cryptococcus neoformans: comparison of two broth microdilution methods andclinical correlates among isolates from Ugandan AIDS patients. J. clin. Microbiol.,36: 2874-2876, 1998.15. JOSEPH-HORNE, T.; HOLLOMON, D.; LOEFFLER, R.S.T. & KELLY, S.L. – Cross-resistance to polyene and azole drug in Cryptococcus neoformans. Antimicrob.Agents Chemother., 39: 1526-1529, 1995.16. JOSEPH-HORNE, T.; LOEFFLER, R.S.T.; HOLLOMON, D.W. & KELLY, S.L. –Amphotericin B resistant isolates of Cryptococcus neoformans without alteration insterol biosynthesis. J. med. vet. Mycol., 34: 223-225, 1996.17. KELLY, S.L.; LAMB, D.C.; TAYLOR, M. et al. - Resistance to amphotericin B associatedwith defective sterol D 8,7 isomerase in a Cryptococcus neoformans strain from AIDSpatient. FEMS Microbiol. Lett., 122: 39-42, 1994.18. KWON-CHUNG, K.J.; POLACHECK, I. & BENNETT, J.E. - Improved medium forseparation of Cryptococcus neoformans var. neoformans (serotypes A and D) andCryptococcus neoformans var. gatti (serotypes B and C). J. clin. Microbiol., 15:535-537, 1982.19. NATIONAL COMMITTEE OF CLINICAL LABORATORY STANDARDS - Referencemethod for broth dilution antifungal susceptibility testing of yeasts: approved standardM27-ª. Wayne, National Committee for Clinical Laboratory Standards, 1997.20. ORNI-WASSERLAUF, R.; IZKHAKOV, E.; SIEGMAN-IGRA, Y. et al. – Fluconazole-resistant Cryptococcus neoformans isolated from an immunocompetent patient withoutprior exposure to fluconazole. Clin. infect. Dis., 29: 1592-1593, 1999.21. PAUGAM, A.; DUPOY-CAMET, J.; BLANCHE, P. et al. – Increased fluconazoleresistance of Cryptococcus neoformans isolated from a patient with AIDS andrecurrent meningitis. Clin. infect. Dis., 19: 975-976, 1994.22. PEETERMANS, W.; BOBBAERS, H.; VERHAEGEN, J. & VANDEPITTE, J. –Fluconazole-resistant Cryptococcus neoformans var. gatti in an AIDS patient. Actaclin. belg., 48: 405-409, 1993.23. PFALLER, M.A.; REX, J.H. & RINALDI, M.G. – Antifungal susceptibility testing:technical advances and potential clinical applications. Clin. infect. Dis., 24: 776-784, 1997.3ALVES, S.H.; OLIVEIRA, L.T.; COSTA, J.M.; LUBECK, I.; CASALI, A.K. & VAINSTEIN, M.H. - In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcusneoformans isolated in Southern of Brazil. Rev. Inst. Med. trop. S. Paulo, 43(5):267-270, 2001.24. POWDERLY, W.G.; KEATH, E.A.; SOKOL-ANDERSON, M. et al. – Amphotericin B-resistant Cryptococcus neoformans in a patient with AIDS. Infect. Dis. clin. Pract.,1: 314-316, 1990.25. POWDERLY, W.G. – Cryptococcal meningitis in HIV-infected patients. Curr. infect.Dis. Rep., 2: 352-357, 2000.26. SAAG, M.S.; CLOU, G.A.; GRAYBILL, J.R. et al. – A comparison of itraconazoleversus fluconazole as maintenance therapy for AIDS-associated cryptococcalmeningitis. National Institute of Allergy and Infectious Diseases Mycoses StudyGroup. Clin. infect. Dis., 28: 291- 296, 1999.27. SANATI, H.; MESSER, S.A.; PFALLER, M.A.; WITT, M. et al. – Multicenter evaluationof broth microdilution method for susceptibility testing of Cryptococcus neoformansagainst fluconazole. J. clin. Microbiol., 34: 1280-1282, 1996.28. WITT, M.D.; LEWIS, R.J.; LARSEN, R.A.; MILEFCHIK, E.N. et al – Identification ofpatients with acute AIDS-associated cryptococcal meningitis who can be effectivelytreated with fluconazole: the role of antifungal susceptibility testing. Clin. infect.Dis., 22: 322-328, 1996.Received: 15 February 2001Accepted: 22 August 2001

In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil

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Suscetibilidade in vitro a antifúngicos, de amostras clínicas e ambientais de Cryptococcus neoformans isoladas no sul do Brasil

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