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Mechanisms and clinical significance of antifungal resistance
International Journal of Antimicrobial Agents 16 (2000) 331 – 333 www.ischemo.org
Mechanisms and clinical significance of antifungal resistance J. Bille * Clinical Microbiology Laboratory, Uni6ersity Hospital (CHUV), 1011 Lausanne, Switzerland
Keywords: Polyene resistance; 5-FC resistance; Azole resistance
For fungal as well as bacterial infections, treatment failure — often called clinical resistance — should be clearly distinguished from microbial resistance. The former can be due to many causes pertaining to the host, the offender or the therapy itself, whereas the latter usually is due either to a native or intrinsic state of in vitro resistance, or to the development de novo or the expression of an acquired mechanism of resistance during therapy. Only microbial resistance will be considered here. In vitro determination of susceptibility or resistance to antifungal agents is still far behind that of antibacterial agents. However, for a few years now a standardised broth dilution method has been available which has the great merit of allowing reproducible results and inter-laboratory comparisons [1]. An increase in the number and spectrum of fungal infections, boosted by the AIDs pandemic and advances in anticancer chemotherapy and transplantation medicine has attracted new interest in the development of new compounds with antifungal activity. However, only three families of antifungal agents are still commonly used today; they are polyenes (amphotericin B), 5-fluorocytosine and azole derivatives (especially fluconazole and itraconazole). In vitro intrinsic resistance to amphotericin B is still a rare event, described mainly in some species of Candida (C. lusitaniae, C. guillermondii, and in some molds (Fusarium spp. P. boydii ). It is important to note that the current in vitro testing methodology [1] may underestimate the number of strains resistant or of reduced susceptibility to amphotericin B. Cases of documented acquired resistance to amphotericin B are even more limited up to now, having been described essentially with Cryptococcus neoformans. * Tel.: +41-21-3144057; fax: + 41-21-3144060. E-mail address: [email protected] (J. Bille).
The clinical significance of in vitro resistance to amphotericin B is poorly documented, but a recent observational study of Candida spp bloodstream infections by Clancy and Nguyen suggests that the risk of failure is higher in patients infected with Candida strains having a MIC for amphotericin B higher than 0.38 mg/l at 48 h. rather than a lower value (56% failure rate vs. 16%, rr:3.5) [2]. For 5 FC, the picture is very different. Primary or intrinsic resistance is common in C. albicans (about 10% of the clinical isolates being natively resistant), and is known among Aspergillus spp., C. neoformans, and non-albicans Candida. This resistance is attributed to a defect in cytosine deaminase. Acquired resistance during treatment is so common (at least 30% of C. albicans) that monotherapy with 5 FC is strongly not recommended. Resistance to azole compounds is of high interest for two main reasons. One is the number and importance of yeasts (C. krusei, C. glabrata to some extent) and fungi (in particular Aspergillus spp and fluconazole) which are intrinsically not susceptible to these antifungal drugs. The second consists of acquired resistance to azoles, mainly in C. albicans. This phenomenon has been observed with an increasing frequency, particularly among HIV infected and AIDs patients suffering from oropharyngeal candidiasis and treated by recurrent courses of azoles. This acquired resistance has been correlated to a previous exposure to azoles and advanced immunosuppression and especially to a cumulative dose of fluconazole exceeding 10 g [3]. Before the availability of HAART treatment, clinical and microbiological failure of oral or oesophageal candidiasis was a frequent (up to 30%) problem. The correlation between in vitro resistance and in vivo clinical failure was well documented. Since the advent of HAART therapy, both the incidence of oropharyngeal candidiasis and that of azole resistant C. albicans episodes have dropped [4].
0924-8579/00/$ - $20 © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 2 6 0 - 0
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J. Bille / International Journal of Antimicrobial Agents 16 (2000) 331–333
In other settings such as nosocomial candidaemia, invasive candidiasis in ICU or haemato — oncological patients, the problem of acquired resistance to azole among intrinsically susceptible yeasts is either still limited or not determined. Of concern, however, is the shift observed in many institutions between the proportion of C. albicans and non-albicans among the aetiologic agents of candidaemia. For example, Nguyen et al. observed in a prospective multicentre study of 427 patients with positive blood cultures, a significant increase of non-albicans Candida species, and a significant proportion of breakthrough candidaemia where the organism had a high MIC for the antifungal agent administered [5]. This shift towards an increasing proportion of less susceptible Candida species to azoles has been ascribed to the (over)use of azoles, although no direct proof has been firmly established. In clinical settings other than oropharyngeal candidiasis — candidaemic episodes for example — it has not been possible to establish a clear-cut correlation between in vitro resistance and in vivo failure to azole therapy in large clinical trials [6]. This is certainly due to the heterogeneity of the patients and to many confounding factors such as the presence and severity of underlying diseases and of foreign bodies. However, some selected well-studied cases have been published recently, in which clinical failure or death can be clearly ascribed to the development of in vivo resistance under therapy of a previously susceptible C. albicans strain [7]. Mechanisms of resistance to azoles among Candida species isolates has been a matter of intense investigation over the last 5 years. At least four mechanisms have been discovered, acting often in combination. The most important is a failure to accumulate azoles inside the cells to reach its target, the cytochrome P 450 involved in the biosynthesis pathway of ergosterol. This type of resistance is caused by an active efflux mechanism mediated by two types of multidrug efflux transporters (MDR), the ATP binding cassette (ABC) transporters and the major facilitators (MF). More than 13 of the former (CDR genes) and more than 28 of the latter (MDR genes) have been characterised so far. Evidence of their role in acquired resistance in clinical isolates has been provided by accumulation assay, measure of increased mRNA levels, complementation of Saccharomyces cere6isiae mutant restoring the resistance, and genetic deletion of both alleles in C. albicans resulting in an hyper-susceptible strain [8]. Efflux pumps for azoles have been found in many species of Candida (C. albicans, C. glabrata, C. tropicalis, C. dubliniensis). Beside this major mechanism of resistance, mutations of the target enzyme (Cyt. P 450) have also been found in clinical isolates, conferring various degrees of (cross-)resistance to azoles [9]. Other
mechanisms such as over-expression of the target enzyme or alteration in the ergosterol biosynthetic pathway (other than Cyt. P 450) have been described, mostly on laboratory strains, but they seem to play a minor role [10,11]. Interestingly, some of these mechanisms (efflux in particular) appear to be reversible when the selection pressure is suppressed. New therapeutic modalities have already been tested with success in vitro and in animal model to overcome efflux-mediated resistance by combining efflux blocking agents to azoles [12]. Thus the gap of knowledge between antibacterial and antifungal resistance is rapidly filling, and this is good news in view of the expanding domain of fungal infections. References [1] NCCLS. 1997. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27-A. National Committee for Clinical and Laboratory Standards. Vol 17: No. 9. [2] Clancy CJ, Nguyen MH. Correlation between in vitro susceptibility determined by E test and response to therapy with amphotericin B: results from a multicenter prospective study of candidemia. Antimicrob Agents Chemother 1999;43:1289– 90. [3] Troillet N, Durussel C, Bille J, Glauser MP, Chave JP. Correlation between in vitro susceptibility of Candida albicans and fluconazole-resistant oropharyngeal candidiasis in HIV-infected patients. Eur J Clin Microbiol Infect Dis 1993;12:911– 5. [4] Martins MD, Lozano-Chiu M, Rex JH. Declining rates of oropharyngeal candidiasis and carriage of Candida albicans associated with trends toward reduced rates of carriage of fluconazole-resistant Candida albicans in human immunodeficiency virus-infected patients. Clin Infect Dis 1998;27:1291 – 4. [5] Nguyen MH, Peacock JE, Morris AJ, et al. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617 – 23. [6] The Candidemia Study Group, and the National Institute of Allergy and Infectious Diseases Mycoses Study Group, Rex JH, Bennett JE, Sugar AM, et al. A randomized trial comparing fluconazole with amphotericin B for the treatment of candidemia in patients without neutropenia. New Engl J Med 1994;331:1325– 30. [7] Marr KA, Lyons CN, Rustad T, Bowden RA, White TC. Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother 1998;42:2584 – 9. [8] Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 1995;39:2378– 86. [9] Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14a-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother 1998;42:241 – 53. [10] Sanglard D, Ischer F, Calabrese D, de Micheli M, Bille J. Multiple resistance mechanisms to azole antifungals in yeast clinical isolates. Drug Resist Updates 1998;1:255 – 65.
J. Bille / International Journal of Antimicrobial Agents 16 (2000) 331–333 [11] White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998;11:382–402. [12] Marchetti O, Entenza JM, Sanglard D, Bille J, Glauser MP,
.
333
Moreillon P. Successful treatment of experimental endocarditis due to Candida albicans with the fungicidal combination of fluconazole and cyclosporin A. ICAAC 1998 ( c15006), San Diego, USA.
Mechanisms and clinical significance of antifungal resistance J. Bille * Clinical Microbiology Laboratory, Uni6ersity Hospital (CHUV), 1011 Lausanne, Switzerland
Keywords: Polyene resistance; 5-FC resistance; Azole resistance
For fungal as well as bacterial infections, treatment failure — often called clinical resistance — should be clearly distinguished from microbial resistance. The former can be due to many causes pertaining to the host, the offender or the therapy itself, whereas the latter usually is due either to a native or intrinsic state of in vitro resistance, or to the development de novo or the expression of an acquired mechanism of resistance during therapy. Only microbial resistance will be considered here. In vitro determination of susceptibility or resistance to antifungal agents is still far behind that of antibacterial agents. However, for a few years now a standardised broth dilution method has been available which has the great merit of allowing reproducible results and inter-laboratory comparisons [1]. An increase in the number and spectrum of fungal infections, boosted by the AIDs pandemic and advances in anticancer chemotherapy and transplantation medicine has attracted new interest in the development of new compounds with antifungal activity. However, only three families of antifungal agents are still commonly used today; they are polyenes (amphotericin B), 5-fluorocytosine and azole derivatives (especially fluconazole and itraconazole). In vitro intrinsic resistance to amphotericin B is still a rare event, described mainly in some species of Candida (C. lusitaniae, C. guillermondii, and in some molds (Fusarium spp. P. boydii ). It is important to note that the current in vitro testing methodology [1] may underestimate the number of strains resistant or of reduced susceptibility to amphotericin B. Cases of documented acquired resistance to amphotericin B are even more limited up to now, having been described essentially with Cryptococcus neoformans. * Tel.: +41-21-3144057; fax: + 41-21-3144060. E-mail address: [email protected] (J. Bille).
The clinical significance of in vitro resistance to amphotericin B is poorly documented, but a recent observational study of Candida spp bloodstream infections by Clancy and Nguyen suggests that the risk of failure is higher in patients infected with Candida strains having a MIC for amphotericin B higher than 0.38 mg/l at 48 h. rather than a lower value (56% failure rate vs. 16%, rr:3.5) [2]. For 5 FC, the picture is very different. Primary or intrinsic resistance is common in C. albicans (about 10% of the clinical isolates being natively resistant), and is known among Aspergillus spp., C. neoformans, and non-albicans Candida. This resistance is attributed to a defect in cytosine deaminase. Acquired resistance during treatment is so common (at least 30% of C. albicans) that monotherapy with 5 FC is strongly not recommended. Resistance to azole compounds is of high interest for two main reasons. One is the number and importance of yeasts (C. krusei, C. glabrata to some extent) and fungi (in particular Aspergillus spp and fluconazole) which are intrinsically not susceptible to these antifungal drugs. The second consists of acquired resistance to azoles, mainly in C. albicans. This phenomenon has been observed with an increasing frequency, particularly among HIV infected and AIDs patients suffering from oropharyngeal candidiasis and treated by recurrent courses of azoles. This acquired resistance has been correlated to a previous exposure to azoles and advanced immunosuppression and especially to a cumulative dose of fluconazole exceeding 10 g [3]. Before the availability of HAART treatment, clinical and microbiological failure of oral or oesophageal candidiasis was a frequent (up to 30%) problem. The correlation between in vitro resistance and in vivo clinical failure was well documented. Since the advent of HAART therapy, both the incidence of oropharyngeal candidiasis and that of azole resistant C. albicans episodes have dropped [4].
0924-8579/00/$ - $20 © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 2 6 0 - 0
332
J. Bille / International Journal of Antimicrobial Agents 16 (2000) 331–333
In other settings such as nosocomial candidaemia, invasive candidiasis in ICU or haemato — oncological patients, the problem of acquired resistance to azole among intrinsically susceptible yeasts is either still limited or not determined. Of concern, however, is the shift observed in many institutions between the proportion of C. albicans and non-albicans among the aetiologic agents of candidaemia. For example, Nguyen et al. observed in a prospective multicentre study of 427 patients with positive blood cultures, a significant increase of non-albicans Candida species, and a significant proportion of breakthrough candidaemia where the organism had a high MIC for the antifungal agent administered [5]. This shift towards an increasing proportion of less susceptible Candida species to azoles has been ascribed to the (over)use of azoles, although no direct proof has been firmly established. In clinical settings other than oropharyngeal candidiasis — candidaemic episodes for example — it has not been possible to establish a clear-cut correlation between in vitro resistance and in vivo failure to azole therapy in large clinical trials [6]. This is certainly due to the heterogeneity of the patients and to many confounding factors such as the presence and severity of underlying diseases and of foreign bodies. However, some selected well-studied cases have been published recently, in which clinical failure or death can be clearly ascribed to the development of in vivo resistance under therapy of a previously susceptible C. albicans strain [7]. Mechanisms of resistance to azoles among Candida species isolates has been a matter of intense investigation over the last 5 years. At least four mechanisms have been discovered, acting often in combination. The most important is a failure to accumulate azoles inside the cells to reach its target, the cytochrome P 450 involved in the biosynthesis pathway of ergosterol. This type of resistance is caused by an active efflux mechanism mediated by two types of multidrug efflux transporters (MDR), the ATP binding cassette (ABC) transporters and the major facilitators (MF). More than 13 of the former (CDR genes) and more than 28 of the latter (MDR genes) have been characterised so far. Evidence of their role in acquired resistance in clinical isolates has been provided by accumulation assay, measure of increased mRNA levels, complementation of Saccharomyces cere6isiae mutant restoring the resistance, and genetic deletion of both alleles in C. albicans resulting in an hyper-susceptible strain [8]. Efflux pumps for azoles have been found in many species of Candida (C. albicans, C. glabrata, C. tropicalis, C. dubliniensis). Beside this major mechanism of resistance, mutations of the target enzyme (Cyt. P 450) have also been found in clinical isolates, conferring various degrees of (cross-)resistance to azoles [9]. Other
mechanisms such as over-expression of the target enzyme or alteration in the ergosterol biosynthetic pathway (other than Cyt. P 450) have been described, mostly on laboratory strains, but they seem to play a minor role [10,11]. Interestingly, some of these mechanisms (efflux in particular) appear to be reversible when the selection pressure is suppressed. New therapeutic modalities have already been tested with success in vitro and in animal model to overcome efflux-mediated resistance by combining efflux blocking agents to azoles [12]. Thus the gap of knowledge between antibacterial and antifungal resistance is rapidly filling, and this is good news in view of the expanding domain of fungal infections. References [1] NCCLS. 1997. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27-A. National Committee for Clinical and Laboratory Standards. Vol 17: No. 9. [2] Clancy CJ, Nguyen MH. Correlation between in vitro susceptibility determined by E test and response to therapy with amphotericin B: results from a multicenter prospective study of candidemia. Antimicrob Agents Chemother 1999;43:1289– 90. [3] Troillet N, Durussel C, Bille J, Glauser MP, Chave JP. Correlation between in vitro susceptibility of Candida albicans and fluconazole-resistant oropharyngeal candidiasis in HIV-infected patients. Eur J Clin Microbiol Infect Dis 1993;12:911– 5. [4] Martins MD, Lozano-Chiu M, Rex JH. Declining rates of oropharyngeal candidiasis and carriage of Candida albicans associated with trends toward reduced rates of carriage of fluconazole-resistant Candida albicans in human immunodeficiency virus-infected patients. Clin Infect Dis 1998;27:1291 – 4. [5] Nguyen MH, Peacock JE, Morris AJ, et al. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617 – 23. [6] The Candidemia Study Group, and the National Institute of Allergy and Infectious Diseases Mycoses Study Group, Rex JH, Bennett JE, Sugar AM, et al. A randomized trial comparing fluconazole with amphotericin B for the treatment of candidemia in patients without neutropenia. New Engl J Med 1994;331:1325– 30. [7] Marr KA, Lyons CN, Rustad T, Bowden RA, White TC. Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother 1998;42:2584 – 9. [8] Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 1995;39:2378– 86. [9] Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14a-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother 1998;42:241 – 53. [10] Sanglard D, Ischer F, Calabrese D, de Micheli M, Bille J. Multiple resistance mechanisms to azole antifungals in yeast clinical isolates. Drug Resist Updates 1998;1:255 – 65.
J. Bille / International Journal of Antimicrobial Agents 16 (2000) 331–333 [11] White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998;11:382–402. [12] Marchetti O, Entenza JM, Sanglard D, Bille J, Glauser MP,
.
333
Moreillon P. Successful treatment of experimental endocarditis due to Candida albicans with the fungicidal combination of fluconazole and cyclosporin A. ICAAC 1998 ( c15006), San Diego, USA.