Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University,1101 E. Marshall Street, Richmond, Virginia 23298-0049, USA
The incidence of fungal and yeast infections, especially Candida and Aspergillus, as well as other newer fungal infections has increased considerably in recent years. Treatment failures are due to microbiological or clinical resistance, the latter being related to the drug, host factors, the fungus and the therapeutic procedures. An overview of efforts to find correlations between microbiological and clinical resistance is presented. The NCCLS M27-A consensus document for in vitro susceptibility testing of Candida and Cryptococcus is a good attempt at this, as is the M38-P for some filamentous fungi. The data available thus far indicate that there is a relationship between in vitro resistance and clinical failure, but not between in vitro susceptibility and therapeutic success. Furthermore, the breakpoints (MIC) that can be applied to the susceptibility tests are established based more on the resistance limits than on susceptibility. MIC data are also essential to obtain distribution profiles of MIC values for fungal populations and for future correlations of MICs with clinical response.
Key words: Antifungal - Fungal infections - Susceptibility tests
Utilidad clínica de las pruebas de sensibilidad in vitro a los antifúngicos
La incidencia de infecciones por hongos y levaduras, sobre todo Candida y Aspergillus, así como por otros hongos emergentes, se ha incrementado notablemente en los últimos años. Los fallos del tratamiento se deben a resistencia microbiológica o resistencia clínica, esta última relacionada con el fármaco, factores del huésped, el hongo causante y procedimientos terapéuticos. Se ha intentado establecer una correlación entre resistencia microbiológica y resistencia clínica. El documento de consenso M27-A del NCCLS para las pruebas de sensibilidad in vitro de Candida y Cryptococcus es una buena aproximación, así como el M38-P para algunos hongos filamentosos. Los datos disponibles hasta ahora señalan una asociación entre resistencia in vitro y fallo clínico, pero no entre sensibilidad in vitro y éxito terapéutico, de ahí que los puntos de corte (CMI) aplicables a las pruebas de sensibilidad se hayan establecido basándose más en los límites de resistencia que de sensibilidad. Se considera que estos datos de CMI son esenciales para establecer sus perfiles de distribución para los diferentes hongos y su futura correlación con la respuesta clínica.
Palabas clave: Antifúngicos - Infecciones fúngicas - Pruebas sensibilidad
Ideally, the ultimate role of antifungal susceptibility testing is to correlate the in vitro result with the in vivo activity and predict the outcome of therapy with an antifungal agent. More importantly, in vitro tests should monitor the development of resistance among a normally susceptible population of fungal pathogens. In order to accomplish these roles and to be clinically useful, in vitro results should be evaluated taking into consideration that host, fungus and drug-related factors may have more value than the in vitro result as predictors of the clinical response to antifungal therapy. This paper presents an overview of these issues and related trends and reviews the few well-documented correlations of the minimum inhibitory concentration (MIC) values with clinical outcome that have been reported.
TRENDS IN MEDICAL MYCOLOGY
Incidence of fungal infections
Several trends have been documented in the field of medical mycology since the 1980s (1). The incidence of both yeast (>50%) and mould infections, especially Candida and Aspergillus infections, has increased considerably. Another trend has been the higher incidence of severe infections caused by new and emerging opportunistic fungal pathogens. Previously, some of these fungi were considered laboratory contaminants. The introduction of modern patient management technologies and therapies and the more aggressive use of chemotherapy have been responsible for the overall higher incidence of the mycoses. These advanced technologies have expanded the number of chemically induced immunosuppressive patients and because these patients survive longer, they are highly susceptible to severe fungal infections. Further, the AIDS pandemic has increased the number of immunocompromised individuals who are also at risk for severe mycoses. In addition, nosocomial infections caused by fungi are more frequent and a higher number (46%) of these infections are caused by species of Candida other than Candida albicans.
New and established antifungal agents
Only nine systemic agents are currently licensed for the treatment of systemic mycoses: the polyene amphotericin B and its three lipid formulations (amphotericin B lipid complex, amphotericin B colloidal dispersion, and a liposomal form of amphotericin B); the imidazoles (miconazole and ketoconazole); the triazoles (fluconazole and itraconazole); and the pyfinfidine synthesis inhibitor flucytosine (5-FC) (1). The polyenes act by binding to ergosterol in the cell membrane, causing osmotic instability and loss of membrane integrity. Therefore, resistance to the polyenes is associated with either an alteration of ergosterol biosynthesis, resulting in decreased ergosterol content (2) or with a disruption of the plasma-membrane phospholipids. The fungistatic azoles (imidazoles and triazoles) inhibit the cytochrome P-450-dependent enzymes, which impairs ergosterol synthesis (3). Resistance to the azoles and polyenes is associated with a change in membrane sterol composition through damage of P-450DM. On the other hand, the fungistatic 5-FC acts as a competitive antimetabolite for uracil, which inhibits the synthesis of yeast RNA. Defects in cytosine permease (responsible for 5-FC uptake) and cytosine deaminase (responsible for the deamination to 5-fluorouracil) are associated with 5-FC resistance. The increased use of antifungal agents resulted in the emergence of resistance to established agents and new antifungals were developed either with a broad spectrum of activity to target resistant fungal pathogens [the triazoles voriconazole, posaconazole (SCH 56592) and ravuconazole (BMS 207147)] or with different targets of activity [the glucan synthesis inhibitors caspofungin (MK-0991) and LY 303366 and the chitin synthase inhibitors nykkomycins]. These new agents are under clinical evaluation.
Therapeutic failure may be the result of microbiological resistance (intrinsic or developed during therapy) or clinical resistance. Clinical resistance is associated with the following: i) drug-related factors (drug pharmacokinetics, dose, penetration, stability, protein binding, drug-drug interactions, and fungistatic nature of the agent used); ii) host-related factors (immune response, severity and site of infection, status of current underlying disease and patient compliance); iii) fungus-related factors (virulence of the infecting organism and its interactions with the host and the therapeutic agent); and iv) patient management-related factors (abscess drainage and presence of intravascular catheter or prosthetic cardiac valve) (1, 4, 5). These factors have an effect on the clinical response to antifungal therapy regardless of the MIC endpoint for the infecting organism.
Microbiological resistance is associated with a fungal pathogen for which an antifungal MIC is higher than average; this is the most common cause of refractory infections. The mechanisms of antifungal resistance of the azoles have been studied extensively in the last few years (6, 9). For fluconazole, antimicrobial resistance is usually the result of several cellular mechanisms of resistance: i) the replacement of the susceptible yeast with a resistant species, either Candida krusei or Candida glabrata; ii) the replacement of a susceptible for a resistant strain; iii) genetic alterations; iv) transient gene expression; and v) the genomic instability within the infecting strain. Variation of MICs for individual colonies can be observed in the latter case. At the molecular level, the mechanisms described below have been documented as the cause of azole resistance in Candida spp. (6-10). Changes in the active efflux mechanisms and accumulation of intracellular drug are regulated by two specific pumps [the adenosine 5'-triphosphate-binding cassette (ABC) transporter CDR genes and the major facilitators (multidrug resistance; MDR)] (1, 6-8). An increased expression of the CDR1 and CDR2 genes has been reported as the most common cause of azole resistance in C. albicans (6, 8) and C. glabrata (7). Overexpression of the MDR gene in a fluconazole-resistant strain has also been reported (1). A lack of or a change in drug penetration due to defects in the permeability of the fungal cell has been associated with sterol and phospholipid content and alteration, which leads to azole resistance (6-8). Resistance of C. krusei and C. albicans has been associated both with low intracellular accumulation of fluconazole in cells and a low affinity of the drug for the enzyme P-450DM (1, 10). Reduced itraconazole accumulation in resistant C. krusei isolates, as compared with that in susceptible strains, has been associated with reduced permeability resulting from an increased sterol content (5, 9). However, reduced accumulation of drug in the fungal cell is more likely mediated by an increased efflux activity.
Clinically, several implications of the different mechanisms of resistance identified should be considered in the management of antifungal therapy because either inadequate doses or dosing schedules can induce the selection of a resistant strain or species (1). The evidence of multiple mechanisms of resistance can preclude the development of a simple and rapid method to evaluate microbiological resistance at the molecular level. On the other hand, because the azoles have similar mechanisms of activity and resistance, replacement of one azole by another may not be useful and azole refractory infections are treated with the more toxic polyenes.
Although amphotericin B has been used frequently to treat fungal infections caused by both yeasts and filamentous fungi, primary resistance or the development of resistance to this antifungal agent has not been a significant problem. However, amphotericin B resistance has been documented for Scedosporium prolificans, Pseudallescheria boydii, Fusarium spp., Scopulariopsis spp., Trichosporon beigelii, Candida lusitaniae, Candida guilliermondii and Candida tropicalis, among others (1). Innate or developed azole resistance is found for C. albicans and Candida dubliniensis (mostly in HIV-associated oropharyngeal infections), C. krusei, Candida inconspicua and Candida norvegensis, and for most moulds against fluconazole. An incidence of 4-35% high 5-FC MICs for certain Candida spp. and Cryptococcus neoformans has been reported since the early 1970s (1).
CORRELATIONS OF IN VITRO RESULTS WITH CLINICAL OUT COME IN YEAST INFECTIONS
NCCLS frameworkfor interpretive breakpoints
Only a modest degree of correlation should be expected between MIC endpoints and clinical response to therapy because the clinical infection is a complex and dynamic biological process that affects the outcome of antifungal therapy. In contrast, the in vitro result is obtained in a relatively simple and well-defined matrix. Therefore, several principles must be considered when interpreting the clinical relevance of MIC endpoints (1, 4, 5). Unlike a drug level, the MIC is not a physical or chemical measurement. Variation in the MIC of a drug for a fungal isolate can be observed when almost any of the testing variables (e.g., the inoculum size, medium formulation, length of incubation, incubation temperature, and method and/or criterion of MIC endpoint determination) are changed. Host factors may have more impact on the infection outcome than the in vitro measurement of susceptibility. Because of these many factors, a low MIC does not necessarily predict clinical success. However, in vitro resistance may identify isolates among a population of susceptible strains that are less likely to respond to a specific antifungal regimen.
The broth dilution tests proposed by the National Committee for Clinical Laboratory Standards (NCCLS) (11) as the standard method were more highly developed by 1997 (12). The NCCLS M27-A document describes both macro- and microdilution methods for the antifungal susceptibility testing of Candida spp. and C. neoformans (12). The improved interlaboratory reproducibility resulting from use of NCCLS guidelines has led to the establishment of interpretive breakpoints for fluconazole and itraconazole.
Fluconazole and itraconazole interpretative breakpoints
Fluconazole breakpoints were established on the basis of fluconazole MICs and clinical responses to fluconazole therapy of 219 AIDS patients who had oropharyngeal candidiasis and of 97 non-neutropenic patients with 99 episodes of bloodstream or visceral candidiasis (4). Analysis of the data indicated a significantly lower clinical success rate (46-73%) when isolates had MICs ³64 µg/ml than for isolates with MICs <32 µg/ml (>78-90% success rate). The doses administered were 100 and 400-800 mg fluconazole/day with expected peak fluconazole levels of approximately 6.7 µg/ml and 20-60 µg/ml, respectively. The itraconazole data included MIC and clinical response data from 316 patients with AIDS treated with 200 mg itraconazole solution/day for oropharyngeal candidiasis (4). Tentative breakpoints for itraconazole were established by considering clinical outcome and its correlation with itraconazole MICs and plasma levels. For example, patients with itraconazole plasma levels of £0.5 µg/ml had a clinical success rate of 44% compared with 65% of those with plasma levels of >0.5 µg/ml for organisms with MICs of ³1.0 µg/ml. Fluconazole and itraconazole MICs for these analyses were determined at 48 h by following the NCCLS macrodilution method (12).
On the basis of the analyses described above, the NCCLS subcommittee identified MICs of ³64 mg fluconazole/ml and MICs of ³1 µg itraconazole/ml as the values that predict an increased probability of therapeutic failure (12). The susceptibility of isolates designated as susceptible-dose dependent (MICs of 16-32 µg fluconazole/ml and 0.25-0.5 µg itraconazole/ml) depends on achievable serum levels with doses of >800 mg fluconazole/day or itraconazole serum concentrations of >0.5 µg/ml. Susceptible isolates have MICs of £0.12-8 µg/fluconazole/ml and £0.03-0.12 µg itraconazole/ ml. Results of other studies (5) including correlation studies by using E-test MICs (13) appear to be in agreement with the NCCLS proposed resistance breakpoints for fluconazole and itraconazole in oropharyngeal infections. Monitoring of itraconazole serum concentrations is recommended when the patient is not responding to therapy with this agent. In bloodstream infections, the presence of a central catheter appears to be a predictor of fungemia as well as the main cause of failure of antifungal therapy to clear the bloodstream. Consensus is needed regarding the use of 24 vs. 48 h of incubation to determine azole MICs for Candida species, especially for C. albicans. The 24-h incubation time is more practical and could be more clinically relevant, but clinical correlations should elucidate this issue.
Fluconazole versus nonoropharyngeal yeast infections
Few reports correlating in vitro fluconazole data and clinical response in bloodstream, organ, or deep-seated fungal infections are available (5). Therapeutic failures with fluconazole have been reported for a variety of candidal infections including meningitis, hepatic and urinary tract infections, and symptomatic vulvovaginitis when fluconazole MICs for the infecting isolate were >32 µg/ml. However, some of these patients were receiving <200 mg fluconazole/day. On the basis of available fluconazole breakpoints, the NCCLS subcommittee has not recommended interpretations of C. neoformans susceptibility. However, some correlation has been reported in three studies (14- 16). In one study (14), the increased probability of failure (from <10 to 90%) for patients receiving 800-2,000 mg fluconazole/day was a function of the increase in MIC (from <0.12 to 16 µg/ml), concomitant use of 5-FC, and the result of the first CSF culture. In the second study (15), when blood cultures were positive, the median time for obtaining the first negative culture was 56 days when fluconazole MICs were 4.0 µg/ml and 16 days when MICs were <4.0 µg/ml. In the third study (16), fluconazole MICs >16 µg/ml were associated with relapses for four out of 25 patients receiving 400 mg fluconazole/day as a maintenance dose. Historically, MICs for C. neoformans isolates have been useful in monitoring the potential development of resistance during therapy or in identifying primary resistance when the patient is not responding to an established therapeutic regimen.
5-FC interpretive breakpoints
Before the NCCLS documents became available (11, 12), 5-FC resistance was defined by in vitro data (MICs >12 µg/ ml), drug levels in blood, animal studies (17), and therapeutic failure as a consequence of drug resistance developing during therapy. Because in vitro data for 5-FC by the NCCLS methods were not forthcoming, the NCCLS subcommittee established interpretive breakpoints on the basis of historical data and the drug's pharmacokinetics for Candida spp. (12): strains for which MICs are ³32 µg/ml are considered resistant and strains for which MICs are £4 µg/ml are considered susceptible. The susceptibility of isolates with MICs between 8 and 16 µg/ml is uncertain and data are not available to allow their categorization as either susceptible or resistant.
Amphotericin B versus yeast infections
The NCCLS methods do not appear to distinguish between amphotericin B-resistant and -susceptible yeast isolates, because of the narrow MIC range obtained. Although the use of Antibiotic Medium 3 (M-3), instead of the standard RPMI-1640 broth and of the E-test, could provide more clinically relevant MIC data (12), lot variability and lack of a standardized test medium and format have precluded the establishment of interpretive breakpoints for amphotericin B.
Well-documented reports of the clinical relevance of in vitro resistance are rare for amphotericin B. Twenty-four well-documented cases of amphotericin B resistance (primary or acquired) among Candida spp. were published between 1979 and 1996 (5). These reports included MIC data that were not determined by the NCCLS method and in seven of these reports, the patients were immunocompromised (18). An amphotericin B MIC range from 0.8 to >100 µg/ml was related to clinical resistance (death, failure to clear the involved organ, worsening of the infection, or persistence of the infection), and MICs of <0.8 µg/ml to response to therapy (amphotericin B doses from 0.4-1 mg/ kg/day). In immunocompromised patients similar to those in one of the reports (18), peak plasma levels of 2.4 µg amphotericin B/ml were obtained after intravenous administration of 1 mg amphotericin B/kg and plasma levels of 1.2 µg/ml following a dose of 0.5 mg amphotericin B/kg (18). Thus, serum levels may have been below the MIC value during therapy in some of these patients and the cause of clinical resistance. Development of resistance during treatment with a polyene was documented in three of these reports with an increase in MIC from 0.06 to 4 µg/ml, from 0.31 to >30 µg/ml, and from 1 to >100 µg/ml (5).
It has been reported that either E-test MICs or the minimum fungicidal concentrations (MFCs) are better predictors of clinical failure to amphotericin B therapy than MICs obtained by using either the standard or the M-3 media (19, 20). Therapeutic failure was defined in these two studies of 106 candidemia patients treated with amphotericin B as the persistence of positive blood cultures during therapy (³3 days) or the development of breakthrough candidal fungemia while receiving either empirical or prophylactic amphotericin B for at least 3 days (19). This correlation only included patients from whom the vascular catheter had been removed to avoid the interference of the correlation by this patient management-related factor. These authors proposed 48 h amphotericin B MFCs >1 µg/ml and E-test MICs >0.38 µg/ml as the resistant amphotericin B endpoints or the strongest predictors of microbiological failure (19, 20). However, the methodology for MFC determination has not been standardized and the E-test is available only for investigational purposes.
CORRELATIONS OF IN VITRO RESULTS WITH CLINICAL OUT COME IN MOULD INFECTIONS
Animal and clinical studies
Guidelines have been proposed for the filamentous fungi (moulds) (21), but interpretive breakpoints are not available. The NCCLS M38-P document describes both macro- and microdilution methods for the antifungal susceptibility testing of some common opportunistic moulds. The relevance of in vitro testing using the M38-P guidelines was evaluated in animal test systems for mould infections caused by Aspergillus fumigatus, Aspergillus flavus, P. boydii and Rhizopus arrhizus (22). Some degree of correlation was demonstrated in animals infected with each of two isolates of R. arrhizus isolates susceptible to amphotericin B (MICs <0.5 µg/ml were associated with response) and two Aspergillus spp. isolates susceptible to itraconazole (<1.0 µg/ml were associated with response). In contrast, lack of response was observed with isolates for which MICs were higher (amphotericin B MICs ³2 µg/ml and itraconazole MICs ³ 1.0 µg/ml).
During the past few years, amphotericin B resistance has been associated with MICs >1.0 µg/ml, when tested by nonstandardized methods for disseminated infections caused by Fusarium spp., Aspergillus spp., and S. prolificans (5). Lack of response to treatment in experimental Fusarium infections has been correlated with amphotericin B MICs >1.0 µg/m (5, 23). In these experimental infections, treatment was considered a failure when amphotericin B did not prolong survival or have a significant effect on the fungal burden in the infective tissues. However, since isolates of Fusarium spp. for which MICs are significantly lower are not available, these correlations have not yet been validated. Three strains of itraconazole-resistant A. fumigatus (MICs >16 µg/ml associated with clinical failure) have been described (24). Recently, data from a collaborative study by the NCCLS subcommittee, which included the itraconazole-resistant isolates described above, have revealed that the determination of MICs using the conventional criterion of complete growth inhibition (100% instead of the 50% growth inhibition described in the M38-P document) after 48 h of incubation appear to be the optimal testing conditions to detect potential azole resistance in moulds (25). These refined testing guidelines as well as the results obtained by the evaluation of other species of new and emerging opportunistic fungi will be described in the NCCLS M38-T (to be published by the end of the year 2000). Correlations of in vivo versus in vitro results are needed to assess the clinical utility of MICs for the filamentous fungi. CONCLUSIONS The available NCCLS breakpoints were established on the basis of the principle that antimicrobial resistance should indicate therapeutic failure. The relevance of fluconazole (>64 µg/ml) and itraconazole (>1.0 µg/ml) resistant breakpoints have been confirmed in well-documented cases of treatment failure in oropharyngeal candidal infections in patients with AIDS and HIV-infected persons. Interpretive data obtained in vitro by using a standardized method are needed for other Candida, C. neoformans, and mould infections and for the establishment of interpretive breakpoints for amphotericin B. MIC information can be useful in cryptococcal as well as in candidal infections to monitor possible development of resistance during therapy and to identify primary resistance. These factors are especially important when the patient does not respond to therapy because the type and/or dose of drug can be modified. MIC data are also essential to obtain distribution profiles of MIC values for fungal populations and for future correlations of MICs with clinical response. REFERENCES
During the past few years, amphotericin B resistance has been associated with MICs >1.0 µg/ml, when tested by nonstandardized methods for disseminated infections caused by Fusarium spp., Aspergillus spp., and S. prolificans (5). Lack of response to treatment in experimental Fusarium infections has been correlated with amphotericin B MICs >1.0 µg/m (5, 23). In these experimental infections, treatment was considered a failure when amphotericin B did not prolong survival or have a significant effect on the fungal burden in the infective tissues. However, since isolates of Fusarium spp. for which MICs are significantly lower are not available, these correlations have not yet been validated. Three strains of itraconazole-resistant A. fumigatus (MICs >16 µg/ml associated with clinical failure) have been described (24).
Recently, data from a collaborative study by the NCCLS subcommittee, which included the itraconazole-resistant isolates described above, have revealed that the determination of MICs using the conventional criterion of complete growth inhibition (100% instead of the 50% growth inhibition described in the M38-P document) after 48 h of incubation appear to be the optimal testing conditions to detect potential azole resistance in moulds (25). These refined testing guidelines as well as the results obtained by the evaluation of other species of new and emerging opportunistic fungi will be described in the NCCLS M38-T (to be published by the end of the year 2000). Correlations of in vivo versus in vitro results are needed to assess the clinical utility of MICs for the filamentous fungi.
The available NCCLS breakpoints were established on the basis of the principle that antimicrobial resistance should indicate therapeutic failure. The relevance of fluconazole (>64 µg/ml) and itraconazole (>1.0 µg/ml) resistant breakpoints have been confirmed in well-documented cases of treatment failure in oropharyngeal candidal infections in patients with AIDS and HIV-infected persons. Interpretive data obtained in vitro by using a standardized method are needed for other Candida, C. neoformans, and mould infections and for the establishment of interpretive breakpoints for amphotericin B. MIC information can be useful in cryptococcal as well as in candidal infections to monitor possible development of resistance during therapy and to identify primary resistance. These factors are especially important when the patient does not respond to therapy because the type and/or dose of drug can be modified. MIC data are also essential to obtain distribution profiles of MIC values for fungal populations and for future correlations of MICs with clinical response.