Antifungal Susceptibility Testing

Infect Dis Clin N Am 20 (2006) 699 709 Antifungal Susceptibility Testing Annette W. Fothergill, MA, MBA, MT(ASCP), CLS(NCA) a, Michael G. Rinaldi, PhD a,b, Deanna A. Sutton, PhD, MT, SM(ASCP), SM, RM(NRM) a a Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA b Clinical Microbiology and Mycology Reference Laboratory Merton Minter Drive, Audie L. Murphy Veterans Administration Health System, San Antonio, TX 78229, USA Historically, antifungal susceptibility testing was conducted by various methods that were primarily based on both broth and agar diffusion or disk diffusion procedures. These methods produced results as diverse as the methods themselves. In 1982, the Clinical and Laboratory Standards Institute (CLSI, at that time named the National Committee of Clinical Laboratory Standards, NCCLS) established a subcommittee to review antifungal susceptibility testing. Their findings were documented 3 years later in NCCLS M20-CR (committee report) and stated that approximately 20% of the responding hospital and reference laboratories were indeed conducting antifungal susceptibility testing as part of their patient care program. Testing was predominately by variations of a broth method and was limited to yeast fungi. In addition, the intra-laboratory agreement between laboratories was unacceptable, with results for a given isolate ranging from susceptible to resistant for the same isolate. Following this report the subcommittee determined that it was necessary to develop a standard method for antifungal susceptibility testing. The goal of the subcommittee was to develop a method that was reproducible between laboratories as opposed to developing a method that correlated the minimum inhibitory concentration (MIC) with patient outcome. The subcommittee reported that the E-mail address: fothergill@uthscsa.edu (A.W. Fothergill). 0891-5520/06/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.idc.2006.06.008 id.theclinics.com

700 FOTHERGILL et al standard should be based on a broth method that used a synthetic medium for testing. Yeast testing Following the recruitment of several laboratories from across the United States, a preliminary standard was introduced 7 years following the initial committee report. This standard, M27-P [1], provided guidelines and stipulated the parameters that are still in effect. These parameters include use of RPMI-1640 as the test medium, an inoculum prepared spectrophotometrically to a final test concentration of 0.5 to 2.5 10 3 CFU/ ml, an incubation temperature of 35 C for 48 hours, and the criteria for determining the endpoint or MIC. The MIC is defined as optically clear or the absence of growth for amphotericin B and a 50% reduction in growth for the azoles tested by the microtiter method. Azoles tested by the macrobroth method, however, have endpoints at the concentration at which an 80% reduction in growth occurs. With input from the scientific community, this method has undergone review and has been amended to its current version of M27-A2 [2]. While this method was being developed in the United States, the European community began work on a standard method also. The EUCAST (European Community Antifungal Susceptibility Testing) method, although similar, incorporated some revisions to the CLSI method to include the addition of a higher concentration of glucose to the RPMI-1640. This addition facilitates the rate of fungal growth allowing the MIC to be determined at 24 hours as opposed to the M27-A2 48 hours. Studies have shown that the two methods are equivalent despite these differences [3], and that a given set of isolates can expect the same categoric placement regardless of the method used. Two other methods have evolved from the initial yeast procedure. These methods are described in M38-A for mold testing [4] and M44-A for yeast disk diffusion testing [5]. Realizing that M27-A2 is labor intensive and not practical for busy clinical laboratories, the CLSI introduced M44-A. This method is a disk diffusion method that is similar to the routine Kirby-Bauer method used globally for bacterial susceptibility testing. To date, only fluconazole and voriconazole have been evaluated. This method uses the same Mueller-Hinton agar that is required for bacterial testing but stipulates the addition of methylene blue-glucose to assist with fungal growth and to enhance visualization of the zone diameters. Methylene blue-glucose solution is added to the surface of the Mueller-Hinton agar and is permitted to air-dry before adding the yeast inoculum. Many laboratories find that M44-A fits into their workflow more easily than M27-A2 and is much less costly. Much work has been done to provide quality control limits to ensure this method has the same validity as the original M27-A2 [6].

ANTIFUNGAL SUSCEPTIBILITY TESTING 701 Since approved methods have been developed, the industry has introduced kits to assist laboratories with antifungal susceptibility testing. Systems that have been evaluated include the Yeast One system by Trek Diagnostics and the ETEST by AB Biodisk. These methods are easy to incorporate into the routine laboratory and give equivalent results to M27- A2 [7 9]. In addition, automated methods are under development. Before launching an antifungal susceptibility program, institutions should consider the volume of testing they can expect. The method is inherently variable and reproducibility can be a problem. Another problem is determining who is available to discuss interpretation of the testing. To date, interpretive guidelines are provided only for fluconazole, itraconazole, voriconazole, and 5-fluorocytosine. Unfortunately breakpoints do not exist for amphotericin B or the echinocandins. The interpretive categories for 5-fluorocytosine are the same categories used to interpret bacterial testing. These categories include susceptible (S), intermediate (I), and resistant (R). Azole testing, however, requires a change in these categories. For azoles, the categories include susceptible (S) and resistant (R) with susceptibledose-dependent (SDD) being substituted for the intermediate category. The SDD category relates to yeast testing only and is not interchangeable with the intermediate category associated with bacterial and 5-fluorocytosine breakpoints. This category is in recognition that yeast susceptibility depends on achieving maximum blood levels. By maintaining blood levels with higher doses of antifungal, an isolate with an SDD endpoint may be successfully treated with a given azole [2]. One main problem with antifungal susceptibility testing is the correlation of the MIC with patient outcome. In an article written by Rex and Pfaller [10], the 90-60 rule is discussed. From this rule, some assumptions can be made between the MIC and patient outcome. This rule states that infections caused by isolates that have MICs considered susceptible respond favorably to appropriate therapy approximately 90% of the time, whereas infections caused by isolates with MICs considered resistant respond favorably approximately 60% of the time. As a result of this, physicians are frequently more interested in determining potential resistance than in determining the susceptibility of a given isolate. Amphotericin B is often held up as the standard to which other antifungals are measured. Amphotericin B is easily tested and acceptable results are obtained for most fungal species. One acceptable deviation from the standard is the use of antibiotic medium 3 (M3) as opposed to RPMI-1640. Isolates tested in RPMI-1640 give amphotericin B MICs that are tightly clustered around 1.0 mg/ml. This does not allow the distinction of susceptible isolates from potentially resistant ones. Antibiotic medium 3 provides a wider distribution of MIC values and isolates with low MICs can easily be distinguished from those with much higher MICs. As a result, clinicians must determine which medium is being used when evaluating results. Another concern documented in M27-A2 regards lot-to-lot variability with

702 FOTHERGILL et al M3. This, however, has not been the authors experience over a 25-year history of antifungal susceptibility testing. Mold testing The M38-A method was released in 2002 to accommodate mold testing. This method is virtually identical to M27-A2 with the exception of the inoculum size. The inoculum size continues to be determined spectrophotometrically but to a higher final test concentration of 1 to 5 10 4 CFU/mL. The guideline provides target percent transmission (%T) readings based on conidial size and these are listed by species. Species such as Aspergillus spp, Paecilomyces spp, and Sporothrix spp are measured at 80%T to 82%T, whereas species with larger conidia, such as Fusarium spp, Rhizopus spp, and Scedosporium spp are standardized to 68%T to 70%T. Although efforts are underway to determine the correct %T for most clinically-significant fungi, the list is not yet complete. When testing other fungi not specifically discussed in the M38-A, laboratories must determine the correct %T through trial and error to achieve the correct final concentration. During the tenure of the M27-A2 committee, it was recognized that the scientific community preferred the microtiter method to the macrobroth method. As a result, the macrobroth method is not discussed in M38-A. This poses a problem when the testing of endemic fungi such as Histoplasma capsulatum, Blastomyces dermatitidis, or Coccidioides immitis is necessary. Mold testing may be conducted by the macrobroth method, because early studies have shown that the two methods are equivalent. Other fungi that may benefit from testing by the macrobroth method are those fungi that grow very slowly. It is difficult to hold microtiter tests longer than 72 hours due to of dehydration. Many of the less frequently encountered fungi may require as long as 120 to 144 hours before growth is detected in the drug-free growth control well. For this reason, isolates that are known to be slow growers should be tested by way of the macrobroth method. Endpoint determination is also much more difficult with molds than with the yeast fungi. Although a reduction in turbidity is easily visualized for the yeast fungi, it is not so easily recognized when testing the molds. Because of the unique growth patterns of the mold fungi in the macrobroth system, one looks for a decrease in volume of growth rather than a reduction in turbidity as for the yeasts. In Aspergillus spp, for example, growth is seen as a cottony clump in the broth. To determine an endpoint, the reader must assess the amount of growth for each concentration and call the endpoint at that concentration that has at least 50% smaller volume of growth. Many individuals are not comfortable with this subjective endpoint determination and prefer to leave mold testing to reference centers.

ANTIFUNGAL SUSCEPTIBILITY TESTING 703 Interpretation of results Perhaps the biggest dilemma facing the clinician is determining how best to use the results obtained by the laboratory. Although breakpoints have not been offered for any drug in M38-A or for amphotericin B in M27- A2, the value most often quoted in the literature as a guideline to potential resistance to amphotericin B is any MIC greater than 1.0 mg/ml [11]. Few isolates have such high MICs when tested against amphotericin B. Notable exceptions include Pseudallescheria boydii (Scedosporium apiospermum), Scedosporium prolificans, Paecilomyces lilacinus, Fusarium spp, and some species of Aspergillus other than A fumigatus, most notably A terreus. All of these species give MIC values that are extremely elevated. One yeast that has been reported to possess resistance to amphotericin B is Candida lusitaniae. Early reports labeled this isolate as amphotericin B-resistant [12,13], but susceptibility studies reveal that less than 10% of C lusitaniae isolates tested have MICs that may be considered resistant (Table 1). This species does, however, possess the ability to develop resistance more readily than other Candida spp. It is more difficult to determine the MIC for azoles than for amphotericin B. The endpoint by the microtiter method is the concentration at which a 50% reduction in turbidity can be visualized. Two problems exist with determining this endpoint visually. A 50% reduction in growth is difficult to discern by eye and results are somewhat subjective. A reduction of only 50% is far more growth than can easily be distinguished from lesser percentages of reduction. The difference is subtle. For this reason, a spectrophotometric plate reader should be considered. The other problem is caused by the static nature of the azoles. The earlier azoles, such as fluconazole and itraconazole, act to inhibit fungi but do not typically possess lethal activity. Because of this static nature, trailing may be observed in yeast fungi. The trailing effect occurs when a distinct reduction is seen at a lower concentration but at a point at which significant growth continues through the highest concentration. This pattern may be misinterpreted as resistance by some, thereby accentuating the need for accurate determination of the percent reduction in growth for correct categoric placement. Some of the newer azoles, such as voriconazole and posaconazole, do seem to possess some cidal activity. Although the azoles as a group possess similar mechanisms of action, they clearly have differing spectra of activity. Fluconazole shows good activity against the yeast fungi but is not the drug of choice for most common mold infections, such as aspergillosis. Most yeast fungi show favorable MIC patterns when tested against fluconazole, but some exceptions do exist. Candida krusei is recognized for intrinsic resistance, and testing against fluconazole is not recommended. Of primary concern is acquired resistance. The most notorious species for acquiring resistance to fluconazole following prolonged therapy are C albicans and C glabrata. It is not as common to

704 FOTHERGILL et al Table 1 Susceptibility trends for selected Candida species (N) MIC 50 (mg/ml) MIC 90 (mg/ml) Candida albicans Amphotericin B (1344) 0.25 0.25 Caspofungin (1188) 0.06 0.125 Fluconazole (2147) 0.25 2.0 Voriconazole (716) %0.015 1.0 Candida glabrata Amphotericin B (985) 0.25 5 Caspofungin (1039) 0.125 25 Fluconazole (1676) 8.0 64 Voriconazole (723) 0.5 2 Candida parapsilosis Amphotericin B (619) 0.125 0.25 Caspofungin (606) 0.25 1.0 Fluconazole (858) 0.5 2.0 Voriconazole (334) 0.03 0.125 Candida tropicalis Amphotericin B (300) 0.25 25 Caspofungin (265) 0.06 06 Fluconazole (452) 1.0 16 Voriconazole (175) 0.06 1 Candida lusitaniae Amphotericin B (130) 0.25 0.5 Caspofungin (88) 0.125 0.25 Fluconazole (197) 0.5 2.0 Voriconazole (67) 0.03 0.25 Candida krusei Amphotericin B (142) 0.25 0.5 Caspofungin (141) 0.25 0.25 Fluconazole (0) a d d Voriconazole (123) 0.5 1.0 Abbreviations: MIC, minimum inhibitory concentration; N, number tested. a M27-A2 does not recommend testing of C Krusei against flyconazole. Selected MIC 50 and MIC 90 in vitro Fungus Testing Laboratory data for the most common yeast species against amphotericin B, caspofungin, fluconazole, and voriconazole. observe resistance in C albicans, but some reports list C glabrata resistance as high as 15% of the species population [14]. Cryptococcus spp are generally susceptible and acquired resistance has not been documented. Itraconazole has activity against yeast and mold fungi. It is useful in treating aspergillosis, blastomycosis, coccidioidomycosis, histoplasmosis, and candidiasis. In addition, itraconazole possesses perhaps the lowest endpoints against the dematiaceous fungi and may be considered the drug of choice for treatment of infections caused by isolates from this group. Some species within this group include Bipolaris spp, Alternaria spp, and Exophiala spp. Any time one is evaluating drugs from the same class, cross-resistance is of concern. Comparison of resistance patterns between

ANTIFUNGAL SUSCEPTIBILITY TESTING 705 itraconazole and fluconazole reveal similar percentages of resistance among Candida species. Voriconazole is the newest approved azole and is noted for its activity against Aspergillus spp, S apiospermum, and Fusarium solani. This is remarkable because S apiospermum and F solani are notoriously resistant to all other antifungal agents. Of note is that voriconazole seems to possess lethal rather than static activity against the aspergilli. Susceptibility patterns for voriconazole against the yeast are similar to itraconazole and fluconazole with a notable exception. There is an extremely low incidence of resistance for C krusei against voriconazole with intrinsic resistance reported for fluconazole and approximately 10% of isolates reported as resistant to itraconazole (A. Fothergill, Fungus Testing Laboratory, unpublished data, 2001 2005). One other azole is of interest and is awaiting approval by the Food and Drug Administration (FDA). Posaconazole is approved in Europe, and FDA action is anticipated in the near future. This agent has a broad spectrum of activity. Clinical trials are underway to assess activity against aspergillosis, candidiasis, fusariosis, coccidioidomycosis, and zygomycosis (mucormycosis). Favorable results against species such as Rhizopus and Mucor show that this drug may provide alternative therapy to amphotericin B for infections caused by this group of fungi that historically has been difficult to treat. Resistance with the yeast fungi has not been observed, but some cross-resistance may be expected to occur. The echinocandins are the newest class of antifungals to be released for use within the United States. Currently available agents include caspofungin, micafungin, and anidulafungin. These agents share excellent activity against Candida and Aspergillus spp [15 19]. This class has not yet been evaluated by the CLSI subcommittee and a standard for testing does not exist. Most yeast studies have involved testing in RPMI-1640 with the MIC being the lowest concentration exhibiting a 50% reduction in turbidity. Alternative reports have tested the echinocandins in M3 with the endpoint being the lowest concentration that is optically clear. Results using the two media are significantly different, with M3 results typically fourfold dilutions lower than those obtained with RPMI-1640. Investigational animal studies do not support the high results seen with RPMI and some researchers feel that the M3 result is more representative of a true MIC. Mold testing requires a different endpoint determination for the echinocandins. The MIC is not an appropriate endpoint; therefore, the minimum effective concentration (MEC) is used. Molds tested against the echinocandins result in visible growth through all concentrations. The growth that is present is abnormal and the effect of the drug is obvious. The MEC is the lowest concentration at which this growth defect is visualized. Resistance has not been widely reported for this class of antifungal. There are, however, a few occurrences of isolates with high MICs. Candida parapsilosis and C guilliermondii are two species with notoriously increased MICs. Some clinical isolates of C albicans and C glabrata exist with elevated

706 FOTHERGILL et al MICs, but it is unclear if these isolates are truly resistant in vivo. Susceptibility data for selected yeast and drugs are presented in Table 1. Combination testing Combination testing is one of the hottest topics discussed by clinicians faced with patients who have refractory fungal disease. Historically the only antifungal combination was amphotericin B plus 5-fluorocytosine. Additionally, amphotericin B plus rifampin was believed to have some value in treating certain infections. These combinations have been replaced by combining amphotericin B with some of the newer antifungal agents. It is not uncommon to test amphotericin B plus azoles or echinocandins. Other combinations include azoles plus echinocandins or azoles plus terbinafine. The azole plus terbinafine combinations have resulted in perhaps the most synergistic results. Perhaps the most sought after information is the potential for antagonism as a result of the combination. Although synergistic activity is desirable, the physician may be most concerned by the potential to treat with two agents whose activity is decreased because of negative antifungal/antifungal interactions. The potential for antagonism is real but has not been proven in humans. Animal studies to date have given conflicting results. Theoretically all drugs tested in combination, with the exception of azoles and amphotericin B, should result in indifferent or possible synergistic results. Because of the mechanisms of action of azoles and amphotericin B, however, there may be antagonism when combining these drugs. Although antagonism may not be noted initially, it should only be expressed once inhibition of the 14 alpha-demethylase by the azole results in depletion of ergosterol in the fungal cell wall, thus eliminating the target for amphotericin B. Because staggered introduction is required to detect it, evaluation using checkerboard methods may not detect antagonism. As a result, clinicians are discouraged from concomitant use of azoles and amphotericin B. A more rational approach may be to initiate use of amphotericin B followed by azole therapy when the patient s condition has stabilized [20]. Currently the best that the laboratory can offer is an estimation of which pathogens are more likely to respond to given antifungals alone and in combination. Combination testing is accomplished in the classic checkerboard dilution scheme. All testing parameters remain the same, including medium, inoculum, and incubation. The checkerboard arrangement places single doubling concentrations of drug A along the X-axis and single concentrations of drug B along the Y-axis. Within the checkerboard, every possible concentration combination of the two drugs is tested. The final result is the lowest concentration of drug A plus the lowest concentration of drug B in which the endpoint criteria are met. Although the result can be determined mathematically, it is still a confusing matter. The MIC of each drug within the combination is expressed as a fraction of each drug alone. The

ANTIFUNGAL SUSCEPTIBILITY TESTING 707 Table 2 Aspergillus spp combination testing results Synergistic Indifferent Antagonistic AMBþRIF A fumigatus 0 10(4) 0 A flavus 0 2 0 A terreus 0 2(1) 0 AMBþCAS A fumigatus 0 25(4) 0 A flavus 0 2(1) 0 A terreus 0 3 0 A niger 0 1 0 AMBþITRA A fumigatus 0 7 1 A flavus 0 2(1) 0 A terreus 0 2 0 Emericilla nidulans 0 1 0 AMBþVORI A fumigatus 0 15(2) 2 A flavus 0 1 3 A terreus 0 2(1) 0 A niger 0 3 0 VORIþCAS A fumigatus 0 14(2) 0 A terreus 0 4(1) 0 A niger 0 3 0 Selected in vitro Fungus Testing Laboratory data for combination testing of aspergili. Numbers in parentheses indicate tests that had lower MICs as a result of the combination, formerly know as additive, as opposed to no change in the MIC as a result of the combination. fractions are then added to arrive at the fractional inhibitory concentration index (FICI) [21]. Synergism is achieved when the FICI is less than 0.6 and antagonism occurs with FICI greater than 1. Indifference is noted when the FICI falls between 0.6 and 0.9. Early writings included another category, but this category has fallen from favor for current reporting. The term additivism is considered to provide a confusing connotation and therefore has been eliminated [22]. An FICI that would have placed results in this category are now considered indifferent. Sample results for aspergilli are presented in Table 2. Summary Antifungal susceptibility testing has been in routine use now for more than 15 years and has become a useful tool for clinicians who are faced with difficult treatment decisions. Although most clinicians order susceptibility testing, much confusion still exists regarding the use of the results. Sufficient data have been generated to determine susceptibility trends for

708 FOTHERGILL et al specific fungi against specific agents, but correlation data are minimal. Despite the lack of correlation data, antifungal susceptibility testing continues to provide useful information to assist with patient care. References [1] NCCLS. Reference method for broth dilution antifungal susceptibility testing of yeasts; proposed standard NCCLS document M27-P. Wayne (PA): National Committee for Clinical Laboratory Standards; 1992. [2] NCCLS. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard NCCLS document M27 A2. Wayne (PA): National Committee for Clinical Laboratory Standards; 2002. [3] Espinel-Ingroff A, Barchiesi F, Cuenca-Estrella M, et al. International and multicenter comparison of EUCAST and CLSI M27 A2 broth microdilution methods for testing susceptibilities of Candida spp to fluconazole, itraconazole, posaconazole, and voriconazole. J Clin Microbiol 2005;43(8):3884 9. [4] NCCLS. Reference method for broth dilution antifungal susceptibility testing of conidialforming filamentous fungi; approved standard NCCLS M38-A. Wayne (PA): National Committee for Clinical Laboratory Standards; 2002. [5] NCCLS. Reference method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline NCCLS document M44-A. Wayne (PA): National Committee for Clinical Laboratory Standards; 2004. [6] Barry A, Bille J, Brown S, et al. Quality control limits from fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol 2003; 41(7):3410 2. [7] Espinel-Ingroff A, Pfaller M, Messer SA, et al. Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the NCCLS M27 A2 reference methods for testing new antifungal agents against clinical isolates of Candida spp. J Clin Microbiol 2004;42(2): 718 21. [8] Maxwell MJ, Messer SA, Hollis RJ, et al. Evaluation of ETEST method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood. J Clin Microbiol 2003;41(3):1087 90. [9] Maxwell MJ, Messer SA, Hollis RJ, et al. Evaluation of ETEST method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans. J Clin Microbiol 2003;41(1):97 9. [10] Rex JH, Pfaller MA. Has antifungal susceptibility testing come of age? Clin Infect Dis 2002; 35(8):982 9. [11] Chaturvedi V, Ramani R, Rex JH. Collaborative study of antibiotic medium 3 and flow cytometry for identification of amphotericin B-resistant Candida isolates. J Clin Microbiol 2004;42(5):2252 4. [12] Pappagianis D, Collins MS, Hector R, et al. Development of resistance to amphotericin B in Candida lusitaniae infecting a human. Antimicrob Agents Chemother 1979;16:123 6. [13] Merz WG. Candida lusitaniae: frequency of recovery, colonization, infection, and amphotericin B resistance. J Clin Microbiol 1984;20:1194 5. [14] Pfaller MA, Messer SA, Hollis RJ, et al. Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States. Diagn Microbiol Infect Dis 1999;33:217 22. [15] Messer SA, Diekema DJ, Boyken L, et al. Activities of micafungin against 315 invasive clinical isolates of fluconazole-resistant Candida spp. J Clin Microbiol 2006;44(2):324 6. [16] Odds FC, Motyl M, Andrade R, et al. Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species. J Clin Microbiol 2004; 42(8):3475 82.

ANTIFUNGAL SUSCEPTIBILITY TESTING 709 [17] Pfaller MA, Boyken L, Hollis RJ, et al. In vitro susceptibilities of Candida spp to caspofungin; four years of global surveillance. J Clin Microbiol 2006;44(3):760 3. [18] Pfaller MA, Boyken L, Hollis RJ, et al. In vitro activities of anidulafungin against more than 2,500 clinical isolates of Candida spp, including 315 isolates resistant to fluconazole. J Clin Microbiol 2005;43(11):5424 7. [19] Pfaller MA, Diekema DJ, Boyken L, et al. Effectiveness of anidulafungin in eradicating Candida species in invasive candidiasis. Antimicrob Agents Chemother 2005;49(11):4795 7. [20] Lewis RE, Kontoyiannis DP. Rationale for combination antifungal therapy. Pharmacotherapy 2001;21(8 Pt 2):149S 64S. [21] Johnson MD, MacDougal C, Ostrosky-Zeichner L, et al. Mini review: combination antifungal therapy. Antimicrob Agents Chemother 2004;48(3):693 715. [22] Greco WR, Bravo G, Parsons JC. The search for synergy; a critical review from a response surface perspective. Pharmacol Rev 1995;47:331 85.