Antibiotics in Laboratory Medicine, 6 Ed.

Chapter 6. Antifungal Drugs: Mechanisms of Action, Drug Resistance, Susceptibility Testing, and Assays of Activity in Biologic Fluids

George R. Thompson III and Thomas F. Patterson

The number of agents available to treat fungal infections has increased by 30% since the year 2000 and several more agents are currently in various stages of clinical development. The greater number of medications now available allows for therapeutic choices; however, differences in antifungal spectrum of activity, bioavailability, formulation, drug interactions, and side effects necessitate a detailed knowledge of each drug class (1).

Despite these advances in antifungal therapy, mortality remains high especially in severely immunocompromised patients. The number of those at risk continues to increase with the greater number of solid organ and bone marrow transplant patients, the use of corticosteroids and other immune-modulating drugs such as tumor necrosis factor alpha (TNF-α) inhibitors, and the epidemic of infection with HIV.

In this chapter, we will review currently available antifungal agents, both systemic and topical (Table 6.1). We will discuss their spectrum, potency, mechanism of action, clinical indications for use, and summarize pharmacokinetic and pharmacodynamic parameters. Furthermore, we will discuss the mechanisms of resistance to the various classes of antifungal agents and the in vitro methods for determining the susceptibility and resistance of fungi to currently available agents. Finally, we will provide an overview of the methods for measuring the concentration of antifungal agents in clinical samples and, where appropriate, discuss the indication for performing such analysis.



Amphotericin B (AmB), natamycin, and nystatin are the currently available polyenes, although differing safety profiles have limited natamycin and nystatin to topical use (2). Polyenes possess a large lactone ring, with a lipophilic chain containing three to seven double bonds and a flexible hydrophilic portion bearing several hydroxyl groups. AmB contains seven conjugated double bonds and may be inactivated by heat, light, and extremes of pH (3). Additionally, AmB is poorly soluble and is not absorbed following oral or intramusclar delivery.

The polyenes bind to ergosterol present within the fungal cell wall membrane. This process disrupts cell wall permeability with the subsequent efflux of potassium and intracellular molecules, causing fungal death due to osmotic instability (4). There is also evidence that AmB acts as a proinflammatory agent and further serves to stimulate innate host immunity. This process involves the interaction of AmB with toll-like receptor 2 (TLR-2), the CD14 receptor, and by stimulating the release of cytokines, chemokines, and other immunologic mediators (5). It has been suggested that AmB may interact with host humoral immunity following the observation of synergistic activity of AmB and antibodies directed at heat shock protein 90 (hsp90), although further confirmatory data is needed (4). These secondary mechanims serve to increase the cascade of oxidative reactions, allowing for the rapid fungicidal activity of AmB formulations.

The spectrum of activity of AmB is broad and includes most strains of CandidaCryptococcus neoformansAspergillus spp, the MucoralesBlastomyces dermatitidisCoccidioides spp, Histoplasma capsulatum, and Paracoccidioides brasiliensisCandida lusitaniaeAspergillus terreusFusarium spp, Pseudallescheria boydiiScedosporium spp, and Trichosporon asahii and certain dematiaceous fungi may be resistant to AmB (69).

When AmB resistance occurs, it is generally attributed to reductions in ergosterol biosynthesis or the synthesis of alternative sterols with a reduced affinity for AMB. However, decreased susceptibility to oxidative damage secondary to increased catalase activity has also been described and may play a secondary role in polyene resistance (10).

Conventional amphotericin B deoxycholate (AmB-d) has been the mainstay of antifungal therapy prior to the development of other antifungal classes; yet the nephrotoxicity of AmB-d was one of the predominant forces driving the pursuit of alternative agents with a diminished side effect profile. Lipid formulations of AmB have subsequently been developed and in most instances have replaced AmB-d (11). The lipid-based formulations of AmB include amphotericin B lipid complex (ABLC), liposomal amphotericin B (L-AmB), and amphotericin B cholesteryl sulfate complex (ABCD) and exhibit a lower incidence of nephrotoxicity than that of AmB-d (12), although the safety profile of ABCD has limited its clinical use.

AmB must be administered parenterally because of its poor solubility and poor absorption when administered orally. It is widely distributed in various tissues and organs, including liver, spleen, bone marrow, kidney, and lung (13). Despite negligible concentration in cerebrospinal fluid (CSF), AmB formulations are effective in treating fungal infections of the central nervous system (CNS). In the past, intrathecal administration of AmB-d was used to treat meningeal infection (14,15); however, this practice is seldom used due to the difficulty of administration, poor patient tolerability, availability of alternative agents, and diminishing physician familiarity with this technique. The tissue distribution of the lipid formulations is similar to that of AmB-d; however, urinary excretion of lipid drugs is lower than that of AmB-d (16). All currently available formulations are highly protein bound (>95%, primarily to albumin) and have long half-lives.

AmB displays concentration-dependent fungicidal activity against many fungi with an optimal maximal concentration-to-mean inhibitory concentration (Cmax-to-MIC) ratio of 4–8:1 and a postantifungal effect of up to 12 hours (17). These findings suggest the peak serum level-to-MIC ratio is the best pharmacologic predictor of outcomes with polyene therapy. Drug levels are infrequently measured nor are necessary and are typically necessary only in the research setting (18).

AmB exhibits poor CSF levels (<5% of concurrent serum concentration); however, this agent remains the treatment of choice for cryptococcal meningitis (19). AmB formulations also have low vitreous penetration (0% to 38%) and intraocular injections may be required to achieve appropriate levels during therapy of deep ophthalmologic fungal infections including candidal endophthalmitis (20,21). The exact route of elimination of AmB is not known and despite the well-known nephrotoxicity, dosing need not be adjusted in patients with a decreased glomerular filtration rate (GFR).

AmB formulations are currently used primarily in the treatment of invasive cryptococcosis and moderate to severe infections with the endemic fungi (HistoplasmaCoccidioidesBlastomyces, etc.). Its use in the treatment of candidiasis and aspergillosis has been largely supplanted by other agents following landmark clinical trials (22,23). L-AmB is also commonly used in the treatment of febrile neutropenia refractory to broad-spectrum antibacterial agents (24). Histoplasmosis and cryptococcosis remain the only infections for which a lipid formulation of AmB (L-AmB) has demonstrated greater efficacy than the conventional form (25,26).

AmB formulations were previously the preferred first-line agent during the treatment of invasive aspergillosis (IA); however, a greater therapeutic response and survival have been demonstrated when voriconazole is administered in this setting—relegating AmB to second-line or salvage therapy during the treatment of IA (27). AmB does remain the agent of choice when Mucorales are encountered. In fact, a delay in the prescribing of an AmB formulation in patients infected with an agent of mucormycosis resulted in a twofold greater risk of death (28). Discriminating between invasive mucormycosis and aspergillosis is difficult, but the differences in the choice of antifungal agents and outcomes mandate an aggressive diagnostic strategy and prompt initiation of antifungal agents.

In attempts to avoid the potential nephrotoxicity of systemic administration and to deliver higher local concentrations, different formulations of AmB have been given via the inhalational route. AmB-d is often difficult to effectively administer in an aerosol form due to foaming caused by the solubilizing agent and the detergent-like effects are thought to possibly affect alveolar surfactant (2831). Thus, lipid preparations are preferred for inhalation delivery. Aerosol delivery has been found effective in the prevention of pulmonary fungal infections in lung transplantation and in bone marrow transplant recipients, although data supporting its efficacy in other settings is limited (29).

Intravenous (IV) infusion of AmB-d is associated with reactions such as fever, chills, rigors, myalgias, bronchospasm, nausea and vomiting, tachycardia, tachypnea, and hypertension (32). These events are less likely to occur when one of the lipid formulations is used; however, ABCD has been associated with the development of dyspnea and hypoxia and L-AmB has been associated with back pain during infusion (20). AmB has been associated with acute kidney injury and nephrotoxicity in many studies and is a well-known potential complication of therapy occurring in up to 30% of patients. This toxicity is thought secondary to vascular smooth muscle dysfunction with resultant vasoconstriction and ischemia (33). For this reason, most advocate ensuring adequate volume status prior to administration. Lipid preparations of AmB have a lower incidence of renal toxicity, and studies have shown that when AmB-d is replaced by a lipid formulation after the development of creatinine elevation, renal function stabilizes or improves in a significant proportion of patients (34).

The avoidance of AmB-d and use of a lipid formulation has been met with skepticism by some due to the price difference in compounds. The reduction in hospital days when toxicity is avoided has proven the lipid formulations more cost-effective than AmB (34).


The antifungal azoles include the imidazoles and the triazoles, which differ in terms of their chemical structure. Among the imidazoles (two nitrogens in the azole ring), only ketoconazole has systemic activity, and this agent has been replaced by more efficacious and less toxic alternatives and thus will not be discussed in this chapter. The triazoles (three nitrogens in the azole ring) all have systemic activity and include fluconazole, itraconazole, voriconazole, and posaconazole. An additional triazole, isavuconazole, is currently in phase III clinical trials.

The triazoles also exert their effects within the fungal cell membrane. The inhibition of cytochrome P450 (CYP)–dependent 14-α-demethylase prevents the conversion of lanosterol to ergosterol. This mechanism results in the accumulation of toxic methylsterols and resultant inhibition of fungal cell growth and replication (Fig. 6.1). This class of agents has demonstrated both species- and strain-dependent fungistatic or fungicidal activity in vitro (35). Generally, these agents exhibit fungistatic activity against yeasts such as Candida and Cryptococcus; however, voriconazole appears to be fungicidal against Aspergillus spp. The area under the curve-to-MIC ratio (AUC/MIC) is the primary predictor of drug efficacy (36).

The indirect immunomodulatory effects are poorly understood due to the complex interaction of triazoles and phagocytic cells. Evidence suggests that ergosterol depletion increases fungal cell vulnerability to phagocytic oxidative damage (37) and voriconazole has been shown to induce the expression of TLR-2, nuclear factor-κB (NF-κB), and TNF-α (4).

Azoles differ in their affinity for the 14-α-demethylase enzyme and this difference is largely responsible for their varying antifungal potency and spectrum of activity. Cross-inhibition of several human CYP-dependent enzymes (3A4, 2C9, and 2C19) is responsible for the majority of the clinical side effects and drug interaction profiles that have been described with this class (3841).

Itraconazole and posaconazole act primarily as inhibitors of 3A4 and 2C9 with little effect on 2C19. However, voriconazole acts as both an inhibitor and a substrate on all three isoenzymes, providing ample opportunity for drug–drug interactions due to this frequently shared metabolic pathway (41).

Comprehensive lists of triazole drug interactions can be found elsewhere (42). Briefly, caution should be used when these agents are concurrently administered with most HMG-CoA reductase inhibitors, benzodiazepines, phenytoin, carbamazepine, cyclosporine, tacrolimus, sirolimus, methylprednisolone, buspirone, alfentanil; the dihydropyridine calcium channel blockers verapamil and diltiazem; the sulfonylureas, rifampin, rifabutin, vincristine, busulfan, docetaxel, trimetrexate; and the protease inhibitors ritonavir, indinavir, and saquinavir (4349).

The triazoles have also been associated with QTc prolongation (50) and coadministration with other agents known to have similar effects (cisapride, terfenadine, astemizole, mizolastine, dofetilide, quinidine, and pimozide, among others) should be avoided (5153). The triazoles are additionally embryotoxic and teratogenic and are secreted into breast milk and thus, administration should be avoided during pregnancy or while lactating (50,54,55).


Fluconazole remains one of the most frequently prescribed triazoles due to its excellent bioavailability, tolerability, and side effect profile. More than 80% of ingested drug is found in the circulation, protein binding is low (approximately 10%), and 60% to 70% is excreted unchanged in the urine (56). Oral absorption remains unchanged in patients receiving acid suppressive therapy (proton pump inhibitors or H2-blockers) (57).

Fluconazole is a water-soluble compound with excellent tissue penetration and is available in both oral and IV formulations. CSF levels are 50% to 70% of matched serum levels, and levels reported in saliva, sputum, and other sites are well within therapeutic ranges (58,59). The half-life is 27 to 34 hours in the presence of normal renal function, allowing once-daily dosing and it exhibits linear pharmacokinetics that are independent of doses and formulation. In patients with diminished creatinine clearance (CrCl), the normal dose should be reduced by 50% (38). Fluconazole exhibits concentration-independent fungistatic activity against Candida and Cryptococcus neoformans (60,61). The AUC/MIC ratio appears to be the most predictive pharmacodynamic parameter for fluconazole (62). Fluconazole serum levels are rarely necessary in clinical practice due to its predictable oral absorption and pharmacokinetic profile.

Fluconazole is active against most Candida spp with the exception of Candida krusei, which is intrinsically resistant to fluconazole (63). Candida glabrata is significantly less susceptible to fluconazole than Candida albicans, although wild-type isolates have MICs that extend to an MIC of more than 64 µg/mL. Previously, Candida glabrata strains were considered susceptible-dose dependent (S-DD at an MIC of 16 to 32 µg/mL), with roughly 10% exhibiting high-level resistance (MIC, more than 64 µg/mL) (21,64). A recent Clinical and Laboratory Standards Institute (CLSI) reclassification has resulted in the breakpoint for susceptible Candida glabrata being eliminated and strains considered S-DD at an MIC of less than or equal to 32 µg/mL. Fluconazole is active against most species of CandidaCryptococcus neoformans, dermatophytes, Trichosporon spp, H. capsulatumCoccidioides immitis, and P. brasiliensis (65,66). Only limited activity is seen against B. dermatitidis and resistance may develop when fluconazole is used to treat histoplasmosis (67). Fluconazole has no useful activity against molds including Aspergillus spp, Fusarium spp, or Mucorales (68).

Fluconazole has an important role in the treatment of candidiasis, cryptococcosis, and coccidioidomycosis (14,69,70). Fluconazole is used as primary therapy for candidemia and mucosal candidiasis, as well as prophylaxis in selected high-risk populations who are at lower risk for molds like Aspergillus (69). Patients with oropharyngeal candidiasis (OPC) are typically prescribed 100 mg/day for 7 to 14 days (21). Newer data suggests a one-time dose of 750 mg for the treatment of OPC with equivalent relapse rates to standard therapy (71). Patients with frequent relapse should remain on chronic suppressive fluconazole until immune reconstitution has been documented.

It is also used in maintenance therapy of cryptococcal meningitis in patients with AIDS (14,72). Following induction therapy with AmB and flucytosine, fluconazole is prescribed as an initial dose of 400 mg for 10 weeks followed by 200 mg weekly pending immune reconstitution (19). Although recent data has accrued regarding the use of high-dose fluconazole monotherapy during the induction course of cryptococcal meningitis, this practice should be used only in resource-limited settings and not when AmB is available (73).

Fluconazole is also the agent of choice in the treatment of disseminated coccidioidomycosis including meningitis. In these cases, high-dose fluconazole (up to 2 g daily) is often necessary (74). Similarly, other endemic mycoses respond favorable to fluconazole and this agent is considered second line during treatment of histoplasmosis and sporotrichosis (75,76).

Fluconazole has also been used for prophylaxis in those at high risk of invasive fungal infections. Initiation of 400 mg/day of fluconazole for the first 75 days following bone marrow transplantation (BMT) has been found effective in reducing cases of candidemia (77). Preemptive therapy in other settings, including within intensive care unit (ICU), remains controversial. The high incidence of invasive candidiasis within this setting (1% to 2% of all patients) makes prophylaxis an attractive option; however, the largest randomized, multicenter, blinded clinical trial comparing empiric fluconazole therapy to placebo in ICU patients with several risk factors for invasive candidiasis showed no clear benefit to fluconazole therapy (78).

Drug–drug interactions are less commonly observed with fluconazole than other triazole compounds, although caution remains necessary due to increases in the serum levels of phenytoin, glipizide, glyburide, warfarin, rifabutin, and cyclosporine. Fluconazole levels are reduced in the presence of rifampin (38).

Fluconazole is well tolerated by most patients, even if chronic therapy is necessary (79). Headache, alopecia, and anorexia are the side effects most common (10%), with transaminase elevation seen in fewer than 10%. Severe side effects such as exfoliative dermatitis and liver failure are very uncommon (38).


Itraconazole is a lipophilic triazole currently available as both capsules and an oral solution suspended in hydroxypropyl-β-cyclodextrin (HP-β-CD) (39). The IV preparation of itraconazole is no longer commercially available.

The IV formulation provides optimal bioavailability with peak plasma levels obtained within 1 hour of administration. The HP-β-CD carrier is cleared renally and caution is recommended when administering the IV formulation to patients with impaired renal function (80). Itraconazole exhibits poor penetration into the CNS and a P-glycoprotein efflux mechanism has been found in mice to actively transport itraconazole out of the brain (81). The high protein binding (less than 1% available as free drug) and lipophilic nature of itraconazole also results in high concentrations in fatty tissues and purulent exudates (82).

Itraconazole exhibits concentration-independent fungistatic activity against Candida spp and Cryptococcus neoformans (83). In contrast, itraconazole has been shown to exert a time- and concentration-dependent fungicidal effect against Aspergillus spp (84).

The antifungal activity of itraconazole is broad and includes Candida spp, Cryptococcus spp, Aspergillus spp, dermatophytes, dematiaceous molds, Pseudallescheria boydiiPenicillium marneffeiB. dermatitidisCoccidioides immitisH. capsulatumParacoccidioides brasiliensis, and Sporothrix schenckii (Table 6.2). Itraconazole has activity against some, but not all, fluconazole-resistant strains of Candida krusei and Candida glabrata. Although rare, strains of Aspergillus fumigatus that are resistant to itraconazole have been reported and may be increasing in some regions (85,86). Mucorales, most strains of Fusarium, and Scedosporium prolificans are resistant to itraconazole (35).

Absorption of itraconazole capsules is erratic and requires an acidic gastric pH and administration with food. Itraconazole solution allows for greater oral bioavailability and the AUC and peak concentrations are both increased by 30% when itraconazole solution is taken in the fasting state (87,88). The cyclodextrin carrier has minimal absorption and no systemic side effects have been attributed to its use in the oral formulation (89). With once-daily dosing, steady state is reached in 7 to 14 days, although oral loading (200 mg three times daily for 3 days) allows for more rapid attainment of therapeutic serum levels (90).

Itraconazole is extensively metabolized by the liver and its major metabolite, hydroxyitraconazole, also possesses antifungal activity similar to that of the parent drug. Despite similar antifungal efficacy, hydroxyitraconazole is not measured during serum drug level determination by high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography with mass spectrometry (UPLC/MS) is instead required. The active metabolite is detected by bioassay (91).

The development of newer and more effective antifungal agents (i.e., voriconazole) has relegated itraconazole to second-line therapy during the treatment of IA. Itraconazole is thus licensed in the United States only for salvage therapy of IA and allergic bronchopulmonary aspergillosis (27).

Itraconazole remains the drug of choice for those with mild to moderate infection caused by histoplasmosis and is the mainstay of secondary prophylaxis in HIV patients with a history of histoplasmosis prior to immune reconstitution with antiretrovirals (75).

It is also useful for lymphocutaneous sporotrichosis and non–life-threatening, nonmeningeal forms of histoplasmosis, paracoccidioidomycosis, and blastomycosis (75,76,9294), and nonmeningeal coccidioidomycosis and is the preferred agent for osseous manifestations of coccidioidomycosis (95). Additionally, itraconazole has been used for maintenance treatment of cryptococcal meningitis (14) and for some forms of phaeohyphomycosis (96). It has no activity against Fusarium spp, Mucorales, or S. prolificans (35).

The recommended dosage of oral itraconazole in adults is 400 mg/day (capsules) and 2.5 mg/kg twice daily (HP-β-CD solution) (27). However, steady-state levels can be more rapidly attained when administered as 200 mg three times daily for 3 days and then 200 mg twice daily for the duration of therapy. Considerable concern remains regarding adequate oral absorption and oral itraconazole is not recommended in seriously ill or patients with life-threatening disease. Dose adjustment is not indicated when the oral formulations of itraconazole is used in patients with renal insufficiency or those receiving hemodialysis/continuous ambulatory peritoneal dialysis (CAPD). The half-life of itraconazole is prolonged in patients with hepatic dysfunction and drug dose adjustment, liver function testing, and drug interactions should be carefully assessed (97).

Side effects due to itraconazole may occur including gastrointestinal intolerance, hypokalemia, edema, rash, and elevated transaminases (39). Severe hepatotoxicity is rare (98). In contrast to fluconazole, drug interactions are relatively common with itraconazole because of the inhibition of the oxidative metabolism of agents that are metabolized by hepatic cytochrome P450 enzymes (39,42).

Itraconazole is usually well tolerated, although adverse reactions have been observed in up to 39% of patients and dose-limited toxicity requiring discontinuation of therapy can occur. The most frequent side effects include nausea and vomiting (<10%), hypertriglyceridemia (9%), hypokalemia (6%), liver enzyme elevations (5%), skin rashes/pruritus (2%), headache and dizziness (<2%), and pedal edema (1%) (99). Gastrointestinal intolerance (46%) is exceedingly common with the oral HP-β-CD solution at doses greater than 400 mg per day, with vomiting as the most frequent complaint (100). The myocardial depressant effects of itraconazole are also well known and cases of congestive heart failure have been reported (101).


Posaconazole is a lipophilic second-generation antifungal triazole with a similar molecular structure to that of itraconazole. However, posaconazole’s improved spectrum of activity also exhibits efficacy against Mucorales and has enhanced activity against Aspergillus spp compared to itraconazole (102).

Posaconazole is insoluble in water and an IV formulation is currently in phase III clinical trials while a solid oral formulation is also currently in development. It is currently administered as a cherry-flavored suspension using polysorbate 80 as the emulsifying agent (103). Optimal dosing of posaconazole is obtained when given as two to four divided doses administered with food or a liquid nutritional supplement (104,105). Although initial studies suggested gastric acidity did not affect posaconazole absorption subsequent work has shown H2-receptor antagonists and proton pump inhibitors may decrease posaconazole serum levels and coadministration should be avoided (54,103,106,107).

Posaconazole has demonstrated dose-dependent pharmacokinetics with saturable absorption above 800 mg per day. Oral loading is thus not possible and steady state is reached only after 7 to 10 days of therapy (108). This prolonged time required to reach steady-state levels also impacts enthusiasm for the use of posaconazole as primary therapy for invasive fungal infections. Posaconazole has a large volume of distribution, despite its high protein binding, and a half-life of approximately 24 hours.

Peak serum concentrations have shown considerable interpatient variability for reasons that remain unclear. Some have proposed genetic polymorphisms within P-glycoprotein play a role as posaconazole is both a substrate and inhibitor, but this remains unproven (109). Glucuronidation plays a minor role in posaconazole metabolism and single-nucleotide polymorphisms within UGT (uridine diphosphate-glucuronosyltransferase) have been proposed to account for these differences but confirmatory studies have not been performed (110). This unpredictable variation in serum posaconazole levels has heightened interest and the necessity of therapeutic drug monitoring (TDM).

Posaconazole is hepatically metabolized and, as discussed earlier, undergoes minimal glucuronidation. Renal clearance plays a minor role in the clearance of posaconazole, which is predominantly eliminated fecally.

Oral posaconazole has proven effective in the prevention of proven or probable IA in neutropenic patients with acute myelogenous leukemia (AML) and in hematopoietic stem cell transplant recipients with graft-versus-host disease (GVHD) (111,112). The efficacy and safety of posaconazole in the treatment of invasive fungal infections has also been assessed, and although this study predates the widespread use of echinocandins and voriconazole, the observed efficacy allows for its use during salvage therapy (113).

Currently, 200 mg three times daily is recommended for prophylaxis, and 800 mg divided in two or four doses is recommended in the salvage setting. Patients not tolerating food should be given a liquid nutritional supplement in attempts to increase drug absorption (109). Pediatric dosing schedules have yet to be definitively established yet retrospective data has demonstrated the safety and efficacy in this population (27,114). Dose adjustment by age, sex, race, and hepatic or renal insufficiency is not necessary given the minimal glucuronidation and renal clearance of posaconazole (115).

Posaconazole is typically well tolerated and infrequently requires discontinuation due to adverse drug events. The most frequent side effects of posaconazole therapy are gastrointestinal (14%), with transaminase elevation and hyperbilirubinemia occurring in 3% (112). However, in one trial, more serious adverse events were reported in patients treated with posaconazole than with fluconazole. Three cardiac events were reported among those possibly related to posaconazole treatment including decreased ejection fraction, QTc prolongation, and torsades de pointes (111). Nevertheless, for most patients, posaconazole is well tolerated and even long-term therapy (>6 months) is frequently without toxicity (116).

Posaconazole is not significantly metabolized through the cytochrome P450 system and serum levels are unlikely to be increased by concomitant administration of P450 inhibitors. Posaconazole is known to decrease the metabolism of other medications metabolized through these pathways and caution should be taken with coadministration.


Voriconazole is a low-molecular-weight water-soluble extended-spectrum triazole with a chemical structure similar to fluconazole. Available in both oral and IV formulations, the latter is dependent on sulfobutyl ether β-cyclodextrin (SBECD) for solubility (117). The oral formulation of voriconazole is well absorbed with a bioavailability of more than 90% (118,119). Steady-state plasma levels range from 2 to 3 µg/mL following oral administration and 3 to 6 µg/mL after IV infusion. When 3 to 6 mg/kg of daily voriconazole is administered, steady-state levels are reached in 5 to 6 days. However, if oral or IV loading is given, steady state can be reached within 1 day (120).

Voriconazole is 58% protein-bound and has excellent penetration into the CNS as well as other tissues (121,122). It is metabolized in the liver via the cytochrome P450 enzyme family and less than 5% of voriconazole is excreted unchanged in urine (41,117).

Although voriconazole in children has demonstrated linear pharmacokinetics, in adults, nonlinear metabolism is observed, likely secondary to saturable metabolic enzymes required for drug clearance (120). Interpatient serum concentration differences have been attributed to polymorphisms within CYP2C19, the major metabolic pathway for voriconazole (117). Up to 20% of non–Indian Asians have low CYP2C19 activity and voriconazole serum levels are thus up to four times higher than those found in White or Black populations in which the “poor metabolizer” status is uncommon (123). The unpredictability of patient enzymatic activity has generated an increased interest in the routine use of voriconazole TDM.

For IV administration, 6 mg/kg twice daily on day 1, followed by 4 mg/kg IV twice daily for the duration of therapy is recommended. The oral dosages in adults are also weight based. For those weighing greater than 40 kg, 400 mg twice daily on day 1, followed by 200 mg twice daily until completion of therapy is suggested, whereas those weighing less than 40 kg should receive 200 mg twice daily for 1 day followed by 100 mg twice daily (123). Pediatric patients are known to hypermetabolize voriconazole and for this reason an IV dose of 7 mg/kg twice daily and oral dosing of 200 mg twice daily without loading is recommended (55). In patients with liver dysfunction, standard loading doses should be given, but the maintenance dose reduced by 50%. The safety of voriconazole use in severe liver disease remains uncertain. No dosage adjustment is required if oral drug is given to patients with renal insufficiency. However, the presence of a cyclodextrin vehicle within the IV formulation has caused concerns about vehicle accumulation in renal insufficiency or dialysis dependence and IV administration is best avoided in patients with a CrCl less than 50 mL (117).

Voriconazole exhibits concentration-independent fungistatic activity against Candida spp and Cryptococcus neoformans with no apparent post–antifungal effect (124). Recent in vivo studies of disseminated Candida albicansinfection indicate that an AUC/MIC ratio of 20 to 25:1 predicts treatment success (125). In contrast, voriconazole appears to exert a dose-dependent fungicidal effect on Aspergillus based on both time-kill studies (126) and clearance of fungal burden in target organs of animal models of IA (127).

Voriconazole has a broad spectrum of activity that includes Candida spp, Cryptococcus neoformansTrichosporon spp, Aspergillus spp, Fusarium spp, and other hyaline molds, dematiaceous fungi, and the endemic fungi. The anticandidal activity of voriconazole encompasses Candida krusei and most, but not all, strains of Candida albicans. Although activity of voriconazole in vitro against Candida glabratacan be demonstrated, the high MICs seen with wild-type Candida glabrata against voriconazole has resulted in no susceptible breakpoints for voriconazole against Candida glabrata in recent CLSI guidance (128,129). Voriconazole is also active against some fungi that are resistant to AmB, including A. terreus and P. boydii (130,131). Voriconazole exhibits a broad spectrum of activity against molds with the exception of agents of mucormycosis (117), and in fact, Mucorales have been proven more virulent if exposed to voriconazole (132).

Voriconazole is approved for the primary treatment of IA (22,133) and for treatment of infections due to P. boydii (Scedosporium apiospermum) and Fusarium spp in patients intolerant of, or with infections refractory to, other antifungal agents (134). Importantly, voriconazole was shown to be more effective than AmB (53% vs. 32% complete or partial response at week 12 of treatment and 71% vs. 58% survival, respectively) for primary treatment of IA (23). Voriconazole has also been shown to have good efficacy in the treatment of various forms of candidiasis (135,136). It has not been approved for empirical treatment of febrile, neutropenic patients despite documented efficacy in the treatment of IA and in the prevention of breakthrough fungal infections in this same patient population (137).

Typically well tolerated, the side effect profile of voriconazole is similar to other triazoles with a few notable exceptions. The majority of those experiencing a reported adverse reaction to voriconazole describe abnormal vision (up to 23%) that is transient, infusion related, and without long-term sequelae (138). This unique effect typically occurs 30 minutes after infusion and abates 30 minutes after onset.

Other well-known effects of voriconazole therapy include skin rash and transaminase elevation in 10% to 20% of recipients (133). Baseline evaluation of hepatic function has been recommended before and during treatment, and rare cases of hepatic failure during voriconazole use have been reported (139). Elevated voriconazole serum levels have been attributed to the majority of side effects encountered in clinical practice, and higher levels (>5.5 mg/L), although associated with favorable outcomes, have also been suggested responsible for the uncommon potential side effects of encephalopathy or hallucinations (140142).

Therapeutic Drug Monitoring for Azole Antifungals

Commercial assays are available for monitoring the serum concentrations of all currently available triazoles; however, at this time, existing guidelines recommend only itraconazole TDM (75,143,144). A strong argument can also be made for routine TDM of both posaconazole and voriconazole as well.

Itraconazole levels should be drawn after steady state is reached to ensure therapeutic levels (>1 µg/mL). Fluconazole levels are infrequently monitored due to the excellent bioavailability of this agent. However, clinical circumstances may dictate drug monitoring when therapeutic levels are uncertain (i.e., concurrent use of rifampin, rifampicin, etc.).

The extended-spectrum triazoles, posaconazole and voriconazole, have received increased attention due to their erratic absorption (posaconazole), or concerns for toxicity and the interpatient variability of serum levels (voriconazole). No guidelines exist for posaconazole TDM; however, past evidence supports a relationship between posaconazole serum drug level and efficacy (145). TDM should also be considered when drug interactions are of concern, such as the aforementioned potential for acid-suppressive agents to reduce absorption, although goal levels remain to be determined. Most experts suggest target trough concentrations greater than or equal to 0.5 µg/mL when given for antifungal prophylaxis. The interpatient variability of voriconazole also warrants consideration of TDM during use. Low concentrations (<1 mg/L) are more common in those receiving oral therapy and failure rates have been associated with low serum drug levels (133). Conversely, levels greater than 5.5 mg/L have been associated with encephalopathy without an improvement in efficacy (140). However, the frequency with which to monitor these newer triazoles remains to be determined.


Echinocandins (caspofungin, micafungin, anidulafungin) are semisynthetic lipopeptides that inhibit the synthesis of β-1,3 glucan, an important constituent of the fungal cell wall (see Fig. 6.1), by inhibiting the activity of glucan synthase. The glucans are important in maintaining the osmotic integrity of the fungal cell and play a key role in cell division and cell growth (1,146). Disruption of β-1,3 glucan synthesis thus impairs cell wall integrity and leads to osmotic lysis (147).

The echinocandins are currently available only in IV formulations. The echinocandins exhibit dose-dependent linear pharmacokinetics and are highly (more than 95%) protein bound (148150). They are broadly distributed to all major organs, including the brain (151), although concentrations in CSF are low (152). Metabolism occurs in the liver by a cytochrome P450 independent mechanism and the inactive metabolites are excreted in the feces and urine; less than 2% of a dose is excreted in the urine in active form (153,154).

Both in vitro and in vivo studies in Candida spp have shown a concentration-dependent fungicidal effect that is optimized at a peak-to-MIC ratio of approximately 8:1 (18,155) and a postantifungal effect of up to 12 hours (156). In contrast, activity against Aspergillus spp is neither classically fungistatic nor fungicidal, with activity localized to the growing hyphal tips and branch points with resultant inhibition of growth and angioinvasion but only a modest effect on the fungal burden in tissues (157159).

TDM of echinocandins is almost never required and not recommended. Administered as standard doses, this class of antifungals has recently received attention for dosing strategies that may be needed in those with morbid obesity, although definitive recommendations have not been made (160,161).

Multiple in vitro studies have confirmed a paradoxical effect of the echinocandins. In this circumstance above a certain concentration of drug decreased antifungal activity is observed. The exact mechanism responsible for this phenomenon has not been fully elucidated, although studies have shown the involvement of the protein kinase C cell wall integrity pathway and an increase in cell wall chitin content as potential mechanisms responsible for this phenomenon (162). The clinical significance of these in vitro findings remains uncertain (163) and higher echinocandin doses have been evaluated in prospective trials and outcomes are no different from those at standard dosing (164).

Echinocandins have poor oral absorption and current agents are available only in the IV formulation. Echinocandins are highly protein bound (anidulafungin 98%, caspofungin 96%, and micafungin 99.8%) and have a half-life of 40 hours, 10.6 hours, and 11 to 17 hours, respectively (80). Their vitreal and CSF penetration is negligible and this point is of clinical significance during the treatment of candidemia if endophthalmitis is also observed (165).

Caspofungin was the first available agent of this class and is metabolized by both hepatic hydrolysis and N-acetylation. Inactive metabolites are subsequently eliminated in the urine. Severe hepatic dysfunction thus mandates caspofungin dose reduction (20). Caspofungin has several drug interactions with agents metabolized through the cytochrome P450 system and serum levels are reduced in the presence of rifampin and may increase levels of sirolimus, nifedipine, and cyclosporine (20). Micafungin is metabolized by nonoxidative metabolism within the liver and anidulafungin undergoes nonenzymatic degradation within the kidney. Both agents are eliminated in feces. These agents therefore do not require dosage adjustment with hepatic impairment (20).

Their clinical use is primarily limited to Candida spp and Aspergillus spp and they lack activity against MucoralesCryptococcus spp, and other clinically important molds (Table 6.1). Although activity is observed against all Candida spp, the mean inhibitory concentrations (MICs) are elevated (>1 µg/mL) when Candida parapsilosis and Candida guilliermondii are encountered. Susceptibility differences between the different agents in this class are minimal (166). Recent changes in CLSI breakpoints recognize that although clinical activity of the echinocandins is good even with higher MICs, mutations in the target FKS1/2 gene are likely present with MICs over 0.25 µg/mL for most species with higher breakpoints for Cparapsilosis and C. guilliermondii. Echinocandins also have immunomodulatory effects. By exposing β-glucan by the disruption of fungal cell wall mannoproteins, additional antigens are exposed for antibody deposition and fungal recognition by the host immune system, which may add activity against organisms such as molds like Mucorales and Fusarium for which they have minimal intrinsic activity (167).

In addition to activity against Candida and Aspergillus spp, the echinocandins have moderate activity against dematiaceous fungi (168,169) and low activity against the endemic dimorphic pathogens (169,170). They are inactive against Fusarium and other hyalohyphomycetes, Cryptococcus neoformansTrichosporum spp, and Mucorales (150,171). Primary resistance to echinocandins appears to be uncommon in Candida albicans and Aspergillus spp and notably, echinocandins are active against fluconazole-resistant strains of Candida spp (32,172,173).

The increased incidence of triazole-resistant Candida spp and the fungicidal activity of the echinocandins (caspofungin, micafungin, and anidulafungin) have prompted some authorities to recommend these agents as first-line therapy for invasive candidiasis and recent meta-analysis has shown improved outcomes in those who receive echinocandins as first-line therapy (174). Additionally, their proven efficacy, infrequency of side effects, and favorable drug interaction profiles make them attractive options over other available antifungals (32,164,175,176).

Comparative trials have found the echinocandins equally efficacious and better tolerated than AmB in the treatment of candidemia (32). In one such trial, caspofungin (70 mg loading dose followed by 50 mg daily) was compared to AmB-d (0.6 to 1 mg/kg) in the treatment of invasive candidiasis. Although C. albicans was more common in the AmB arm, modified intention to treat analysis revealed similar survival in each group, with a trend toward increased survival and a statistically significant decrease in drug side effects in those receiving caspofungin (32).

Similarly, micafungin (100 mg IV daily) has been compared to L-AmB 3 mg/kg IV daily in an international, double-blind trial. In this study assigning patients to 14 days of IV treatment, successful treatment was equivalent in each group. However, there were fewer treatment-related adverse events with micafungin than there were with liposomal amphotericin (175).

Only one comparative trial of different echinocandins has been performed in invasive candidiasis. In this trial, patients were enrolled to one of three treatment groups: micafungin 100 mg/day IV daily, micafungin 150 mg IV daily, or caspofungin 70 mg IV loading dose followed by 50 mg IV daily. No differences were found between treatment groups including microbiologic failure or all-cause mortality (164). Although this trial found that higher doses of an echinocandin may not equate to a greater therapeutic response, no increase in toxicity was seen with higher doses nor was a paradoxical effect observed.

Anidulafungin has been compared with fluconazole for the treatment of invasive candidiasis and treatment was successful in 75.6% of patients treated with anidulafungin, as compared with 60.2% of those treated with fluconazole. Survival was improved with anidulafungin but was not statistically significant compared to the fluconazole arm (176).

Collectively, these trials have shown a high level of efficacy and low toxicity for the echinocandins. This class has since become “first-line” therapy in the treatment of candidiasis in most medical centers pending species identification and documented clinical improvement in the patient. After clinical improvement is obtained or the absence of fluconazole resistance documented, therapy is often changed to a triazole such as fluconazole (69). As noted earlier, CNS and intraocular infections should not be treated with echinocandin monotherapy due to their poor penetration into these sites.

Although clinical trials have been primarily limited to patients with candidemia, observational data has shown efficacy in candidal osteomyelitis, peritoneal infections, and abdominal abscesses (177). Additional retrospective data has also shown echinocandins may play a role in the treatment of infective endocarditis caused by Candida spp (178).

The echinocandins have also been found efficacious in the treatment of IA, although they are fungistatic against this genus. The known toxicity of AmB and its different formulations and the potential for voriconazole-induced drug–drug interactions or toxicity has increased interest in the echinocandins for use during treatment of IA (179).

Caspofungin as a potential first-line agent for IA has been evaluated in limited settings but data is not sufficient to recommend caspofungin or any of the echinocandins for first-line therapy in the treatment of IA. Thus, these agents are currently recommended in the treatment of IA only in those patients refractory to or intolerant of other agents (27,179). In vitro studies and limited clinical data have also shown the potential role for combination therapy (an echinocandin plus AmB or an azole) and a prospective trial of anidulafungin and voriconazole has been completed, with results suggesting benefit in subpopulations of patients but not in the entire population studied, although at the present time, full publication of the trial data has not occurred.

The side-effect profile of the echinocandins is very favorable and these agents are typically well tolerated. The most frequently reported adverse effects include increased liver transaminases, gastrointestinal upset, and headache (32,153). An infusion-related reaction has been described if rapid administration is given with tachycardia, hypotension, and/or thrombophlebitis. Drug interactions are rare; however, concomitant administration of caspofungin and cyclosporine is not recommended, yet reports have shown it may be safe to do so in selected cases (180,181). Cyclosporine has been noted to increase the AUC of caspofungin by approximately 35%, but these alterations are not sufficient to suggest dosage modifications (150). The authors do not typically make dose adjustments of the echinocandins in any of these situations.



Flucytosine (5-FC) is a water-soluble, synthetic, fluorinated pyrimidine analogue that exerts antifungal activity by interfering with the synthesis of DNA, RNA, and proteins in the fungal cell. In vivo, 5-FC is taken up into the cell by a fungus-specific cytosine permease and deaminated in the cytoplasm to 5-fluorouracil (5-FU) by cytosine deaminase. 5-FU is further converted to 5-fluorodeoxyuridylic acid, which interferes with DNA synthesis. Mammalian cells lack cytosine deaminase, allowing for a selective inhibition of fungal organisms (see Fig. 6.1) (182). This agent may be either fungistatic or fungicidal, depending on both fungal species and strain.

5-FC is available in an oral formulation with excellent bioavailability. Unfortunately, the IV formulation is no longer widely available. High concentrations of 5-FC may be achieved in serum, CSF, and other body fluids (183). In vitro pharmacodynamic studies have determined that 5-FC exhibits concentration-independent fungistatic activity against Candida spp that is optimized at concentrations four times the MIC (184). Furthermore, 5-FC exerts a significant post–antifungal effect of 2.5 to 4 hours against Candida spp (184). These findings have been confirmed in vivo by Andes and van Ogtrop (185), who showed that time above the MIC was the pharmacodynamic parameter that correlated best with outcome in 5-FC treatment of murine candidiasis. Maximum efficacy was seen when 5-FC blood levels exceeded the MIC for only 20% to 25% of the dosing interval (185). The later observation may be accounted for by the post–antifungal effect of 5-FC.

5-FC has excellent oral bioavailability with over 80% to 90% absorption. Peak serum levels occur 1 to 2 hours after ingestion (30 to 45 µg/mL) of a single dose. The volume of distribution (Vd) of 5-FC is 0.6 to 0.9, yet bone, peritoneal, and synovial fluid 5-FC levels have been demonstrated and urinary levels are several folds higher than concurrent serum levels. Greater than 95% of 5-FC is eliminated unchanged in the urine. 5-FC is typically administered by mouth at 100 mg/kg/day divided in four doses (80) and that that dose is usually well tolerated without the need to adjust the dose based on drug levels.

Activity has been observed against most fungal pathogens including CandidaCryptococcusCladosporiumPhialophora, and Saccharomyces spp. However, Aspergillus spp, Mucorales, dermatophytes, and the endemic mycoses are all resistant to 5-FC (1). Additionally, resistance commonly develops when 5-FC is used as monotherapy even in susceptible organisms and it should not be used as such except during the treatment of chromoblastomycoses or during the treatment of localized candidal infections when alternative agents are unavailable or contraindicated.

5-FC is primarily used only in the treatment of cryptococcus (combined with AmB) and chromoblastomycosis. Despite concerns for additive toxicity, the synergistic effects of dual therapy in Cryptococcusallow for more rapid CSF clearance (186).

Peak plasma levels of 40 µg/mL easily exceed the MICs of most Candida spp and Cryptococcus neoformans (187,188) and provide optimal antifungal activity while minimizing hematologic adverse effects (189). The marrow suppressive effects are more common if blood levels exceed 100 to 125 µg/mL (190). In the presence of prolonged therapy (>7 days) or with alterations in renal function, serum drug monitoring can be considered but at the lower doses typically recommended for use, drug level monitoring is not frequently necessary. Other less common side effects such as abdominal pain or diarrhea are frequently indirect markers of elevated 5-FC levels and therapy is typically stopped in these circumstances. Rash and hepatic transaminase elevation have also been reported with liver abnormalities frequently observed (183). 5-FC is teratogenic and should not be administered during pregnancy.


The allylamine class of antifungal agents is composed of terbinafine and naftifine. Only terbinafine has systemic activity. Both terbinafine and naftifine inhibit the enzyme squalene epoxidase, which results in the accumulation of squalene and blocks the synthesis of ergosterol (191). The accumulation of high concentrations of squalene results in increased membrane permeability and, ultimately, cell death.


Terbinafine is a lipophilic antifungal agent that is available in oral and topical formulations (192). Terbinafine has a broad spectrum of activity that includes dermatophytes, Candida spp, Malassezia furfurAspergillus spp, Cryptococcus neoformansTrichosporon spp, S. schenckii, and P. marneffei (193196).

Terbinafine is well absorbed orally and is concentrated in fatty tissues, skin, hair, and nails but low serum levels of drug are found (197). Low toxicity and good efficacy in the treatment of virtually all dermatomycoses, including onychomycosis, is seen with terbinafine (198,199). The combination of terbinafine and fluconazole has shown efficacy in the treatment of fluconazole-resistant Candida spp yet with the recent availability of alternative antifungals, other agents are typically chosen (200). Terbinafine also has shown clinical efficacy in cases of sporotrichosis, aspergillosis, and chromoblastomycosis (201).

It is an inhibitor of CYP2D6, but this generally results in less important drug–drug interactions than inhibitors of CYP3A4. The most commonly reported side effects include gastrointestinal upset and cutaneous reactions (202). Reversible agranulocytosis has also been reported but is rare (203,204). Hepatic dysfunction and even fulminant hepatic failure are uncommon but have been reported during terbinafine administration and for this reason, terbinafine is not recommended in those with underlying liver disease (202).


Griseofulvin is a long-standing oral agent used for the treatment of dermatomycoses. Its mechanism of action is thought to involve interaction with microtubules within the fungal cell and inhibition of mitosis (205,206).

Griseofulvin is active against most dermatophytes; however, alternative agents, such as itraconazole and terbinafine, appear to be more potent and exhibit greater efficacy in prospective clinical trials (207210).

Griseofulvin is best absorbed orally when administered in an ultramicrocrystalline form with an accompanying meal. It is deposited primarily in keratin precursor cells. Administration of griseofulvin is associated with a number of mild side effects including nausea, diarrhea, headache, cutaneous disruptions, hepatotoxicity, and neurologic complaints (211).


There are numerous topical antifungal preparations available for the treatment of superficial cutaneous and mucosal fungal infections. The available preparations encompass a wide variety of antifungal classes including polyenes (AmB, nystatin, and natamycin), allylamines (naftifine and terbinafine), and numerous imidazoles and other miscellaneous agents. Preparations for use in cutaneous disease and onychomycosis include creams, lotions, ointments, powders, and sprays, whereas suspensions, tablets, troches, and suppositories are used for treatment of various forms of mucosal candidiasis (69,212,213).

The selection of topical versus systemic therapy for cutaneous or mucosal fungal infections depends on the status of the host and the type and extent of the infection. The refractory nature of infections such as onychomycosis or tinea capitis usually mandates long-term systemic therapy, whereas most cutaneous dermatophytic infections and oral or vaginal thrush respond favorably to topical therapy. Systemic agents are generally recommended for chronic, recurrent oral or vaginal candidiasis and for candidal esophagitis (69,213).


Currently, several different antifungal agents are under active development, including those with established modes of action as well as novel new classes of antifungal agents (Table 6.1). These include a novel triazole agent (isavuconazole), IV and tablet posaconazole formulations, oral echinocandins, a chitin synthase inhibitor (nikkomycin Z), and agents with new mechanisms of action such as blocking critical or unique fungal cell pathways, such as elongation factor, fungal gene methylation, chitin synthesis, and others. The mechanisms of action and spectra of activity of the novel triazoles and the echinocandins are essentially the same as currently available agents. In each case, the newer agents in the respective classes offer the potential of fewer toxicities and drug interactions, more favorable pharmacokinetic and pharmacodynamic properties, and possible improved activity against selected refractory pathogens. Nikkomycin Z, which inhibits chitin synthesis in the fungal cell wall, provides another novel mode of action that may act in concert with other inhibitors of cell wall or cell membrane synthesis. The introduction of several of these new agents such as isavuconazole, which is in active clinical development and the new formulations of posaconazole, should augment our available antifungal resources. The development of agents with novel modes of action is both necessary and promising for future antifungal therapy.


Resistance to antimicrobial agents is an issue of concern worldwide with important implications for morbidity, mortality, and the costs of health care. Although much attention has been focused on antibacterial resistance (214), innate or acquired resistance to antifungal agents is now recognized among several pathogenic fungi (215,219). This recognition has spurred the development of new antifungal agents as well as intensive efforts to define the molecular mechanisms of resistance to various agents (Table 6.3).

The extensive utilization of fluconazole has been coupled with increasing reports of azole resistance (216,217) and some of the most elegant investigations of antifungal resistance mechanisms have involved the azole class of antifungals and Candida spp (218). In this section, we will review what is known of the molecular and cellular mechanisms of resistance to antifungal agents as well as the clinical factors that may result in resistance to antifungal therapy.

Molecular and Cellular Mechanisms of Resistance to Antifungal Agents

Most fungal infections are caused by Candida spp and most of our understanding of the mechanisms of resistance comes from studies of Candida albicans and other species of Candida (218220). Although Aspergillus spp and Cryptococcus neoformans constitute a significant proportion of opportunistic mycoses, fewer studies have been performed on these organisms and little information on antifungal resistance mechanisms is available for other opportunistic fungal pathogens (68).

Although there are many parallels that exist between antibacterial resistance mechanisms and antifungal resistance mechanisms (191), there are no data to suggest that destruction or modification of antifungal agents is an important component of antifungal resistance. Likewise, it does not appear that fungi can employ the genetic exchange mechanisms that allow rapid transmission of antimicrobial resistance in bacteria. On the other hand, it is apparent that multidrug efflux pumps, target alterations, and reduced access to targets are important mechanisms of resistance to antifungal agents, just as they are important in antibacterial resistance (220,221). In contrast to the rapid emergence of high-level antimicrobial resistance that occurs among bacteria, antifungal resistance usually develops slowly and involves the emergence of intrinsically resistant species or a gradual, stepwise alteration of cellular structures or functions that results in resistance to an agent to which there has been prior exposure (221,222).


Resistance to AmB remains uncommon despite extensive utilization over more than 40 years. Among the Candida spp, decreased susceptibility to AmB has been reported in C. lusitaniaeC. glabrataC. krusei, and C. guilliermondii(219,223226). Although primary resistance to AmB is seen chiefly among C. lusitaniaeC. krusei, and C. guilliermondii (68,227,228). Most reports of AmB resistance in Candida spp appear to be secondary to AmB exposure during treatment (219,223).

Most isolates of Aspergillus spp appear susceptible to AmB; however, A. terreus seems to be resistant to AmB both in vitro and in vivo (229,230). Similarly, some of the “cryptic” strains of Aspergillus such as Aspergillus lentulusand others such as Aspergillus calidoustus may show decreased amphotericin susceptibility. Primary resistance to AmB among C. neoformans has not been reported while secondary resistance appears rare (187,231,232).

Our understanding of the mechanism of resistance to AmB stems largely from studies of mutants of Candida spp and Cryptococcus neoformans derived from sequential passage in varying concentrations of AmB (233) and from characterization of serial isolates from patients failing AmB therapy (218,223,231,234238). The mechanism of AmB resistance appears to be from a qualitative or quantitative alteration in the sterol content of cells (239,240), defense mechanisms against oxidative damage (241), defects in ergosterol biosynthetic genes (242), and alterations in the sterol-to-phospholipid ratio (239).

Ergosterol is the primary sterol target for AmB in the fungal cell membrane and resistant yeast containing an altered sterol content bind lesser amounts of AmB than do susceptible cells. Accordingly, mutants of Candida spp and Cryptococcus neoformans resistant to AmB have been shown to have a reduced total ergosterol content (231,235), replacement of polyene-binding sterols (ergosterol) by ones that bind polyenes less well (fecosterol) (231,243), or masking of ergosterol in the cell membrane so that binding with polyenes is hindered by steric or thermodynamic factors (191,244). One or more of these factors may account for decreased susceptibility to AmB. This resistance is often specific for AmB, but cross-resistance to azoles has also been reported and (5,6)-desaturase (Erg3p) mutants identified as causal in isolates with resistance to both classes (223,245). The molecular mechanisms of AmB resistance in Candida and Cryptococcus neoformans have not been determined; however, sterol analyses of resistant isolates suggest that they are defective in erg2 or erg3, genes encoding for the C-8 sterol isomerase and C-5 sterol desaturase enzymes, respectively (218).


The excellent safety profile of fluconazole has led to extensive utilization of this agent worldwide. Concomitant with this utilization reports of emerging resistance to fluconazole and other azoles have appeared (246). Despite these reports, primary resistance to fluconazole is unusual among most species of Candida causing bloodstream infections (216). Among the five most common species of Candidaisolated from blood (C. albicansC. glabrataC. parapsilosisC. tropicalis, and C. krusei), the overall frequency of high-level resistance (previously reported as an MIC >64 µg/mL) to fluconazole is less than 3%. Resistance is uncommon among bloodstream infections of C. albicans (0% to 7%) (216,247), C. parapsilosis (0% to 4%), and C. tropicalis (0% to 6%), whereas approximately 12% or more of C. glabrata isolates exhibit primary resistance to this agent (247,248). C. krusei is considered intrinsically resistant to fluconazole and MICs are usually more than 32 µg/mL for this species (36,249). Notably, the frequency of resistance to fluconazole among bloodstream infection isolates of Candida spp has not increased substantially after more than two decades of utilization worldwide (216,247,250).

The new triazoles (posaconazole and voriconazole) exhibit more potent activity against Candida spp than that of fluconazole, including activity against C. krusei and some fluconazole-resistant strains of Candida (251255). Isolates of C.glabrata for which fluconazole MICs are less than 16 µg/mL typically have MICs less than 1 µg/mL to voriconazole and posaconazole (129,256,257); however, those isolates with higher fluconazole MICs tend to be less susceptible to the new triazoles with MICs more than 2 µg/mL (129,256). Recent breakpoints for voriconazole do not include a susceptible category for voriconazole for C. glabrata and posaconazole breakpoints have not yet been established. In general, among Candida isolates, there is a strong positive correlation between fluconazole MICs and those of voriconazole and posaconazole, suggesting significance cross-resistance, especially for some species such as C. glabrata (129,254).

Similar to Candida spp, primary resistance to fluconazole is uncommon among isolates of Cryptococcus neoformans (187,258,259), although secondary resistance has been described among individuals with AIDS and relapsing cryptococcal meningitis (260). Despite these reports, the susceptibility profile of this organism to fluconazole has remained unchanged over the past 30 years (187,260).

Although Aspergillus spp are intrinsically resistant to fluconazole, most isolates appear susceptible to itraconazole and the new triazoles (261). MICs greater than 1 µg/mL for these azoles are unusual among clinical isolates of Aspergillus, although the incidence may be increasing in certain regions due to environmental use of triazole fungicides (262,263). In contrast to Candida, cross-resistance between itraconazole and the new triazoles is not complete: cross-resistance between itraconazole and posaconazole, but not voriconazole, has been observed in some strains (264).

The mechanism of azole resistance in Candida has been extensively evaluated for fluconazole and C. albicans (218,221). Resistance can result from a modification in the quality or quantity of the target enzyme, reduced access of the drug to the target, or some combination of these mechanisms. In the first instance, point mutations in the gene (ERG11) encoding for the target enzyme, 14-α-demethylase, leads to an altered target with decreased affinity for azoles. Overexpression of ERG11 results in the production of high concentrations of the target enzyme, creating the need for higher intracellular azole concentrations to inhibit increased available substrate. Loss of allelic variation in the ERG11 promoter may also result in a resistant strain that is homozygous for the mutated gene (265).

The second major mechanism involves active efflux of azole antifungal agents out of the cell through the action of two types of multidrug efflux transporters: the major facilitators (encoded by MDR genes) and those of the ATP-binding cassette superfamily (encoded by CDR genes) (219). Upregulation of the MDR1 gene leads to fluconazole resistance, whereas upregulation of CDR genes leads to resistance to multiple azoles (221,266269). Evidence that these mechanisms may act individually, sequentially, and in concert has been derived by studying serial isolates of C. albicans from AIDS patients with OPC (270272).

It appears that the mechanisms of resistance to azoles in C. glabrata involve upregulation of CDR1 genes, resulting in resistance to multiple azoles (266). Azole resistance in C. krusei appears to be mediated by reduced susceptibility of the target enzyme to inhibition by fluconazole and itraconazole (63). This does not seem to be the case with the new triazoles, given their potent aforementioned activity against C. krusei.

In Aspergillus, the azole target cyp51A is an established “hot spot” for mutations that confer phenotypic triazole resistance (273). A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Recently, a mutation in the transcription factor complex subunit HapE has also been reported (274) and with the advent of whole genome sequencing, other mechanisms are likely to be seen in the near future.

The C. neoformans isolates that have developed secondary resistance to fluconazole have been shown to have an altered target enzyme or overexpression of MDR efflux pumps (275,276). The latter mechanism, termed heteroresistance, is intrinsic in all cryptococcal isolates and is easily induced in vitro (277).


Caspofungin, anidulafungin, and micafungin all exhibit potent fungicidal activity against most species of Candida, including azole-resistant strains (18,278,279). Isolates for which echinocandin MICs exceed 1 µg/mL rarely occur outside of C. parapsilosis and C. guilliermondii; however, recent evidence has suggested the incidence of echinocandins resistance in C. glabrata may be increasing (216). Efforts to produce laboratory mutants of Candida spp with reduced susceptibility to caspofungin have demonstrated that the frequency of these mutants is extremely low (1 in 108 cells), suggesting a low potential for the emergence of resistance in the clinical setting (280,281); however, the development of resistance despite continuous receipt of an echinocandins has been reported (282). Likewise, isolates of Aspergillusspp from clinical sources with reduced susceptibility to echinocandins have been observed, although this appears an uncommon occurrence (171,283).

Studies of laboratory-derived mutants of C. albicans with reduced in vitro and in vivo susceptibility to the echinocandins have documented point mutations in the FKS1 and FKS2 genes encoding for the glucan-synthesis enzyme complex (282,284,285). These mutant strains demonstrate an increased 50% inhibitory concentration (IC50) for inhibition of the glucan synthesis enzyme complex and reduced susceptibility to all echinocandins in vitro and in vivo in animal models (280,281,286). These strains remain susceptible to polyenes and azole antifungal agents, although multidrug resistance to both echinocandins and azoles has been reported with C. glabrataFKS genes are similarly essential in Aspergillus spp (287), and increased expression of the FKS gene has been observed in a resistant isolate (283), whereas other strains have been found to have mutations in the ECM33 gene (AfuEcm33), encoding cell wall proteins important for fungal cell wall organization (288,289). Although there are limited data currently, these observations suggest that while mutations in the hot spot regions of the FKS gene is the predominant resistance mechanism in Candida, it is possible that mechanisms outside the target gene may be more important in Aspergillus.


Despite reports in the older literature of a higher frequency of primary resistance to 5-FC among Candida spp and Cryptococcus neoformans (290,291), more recent studies using validated, standardized test methods indicate that primary resistance is actually uncommon among bloodstream infection isolates of both Candida spp and Cryptococcus (187,188). Secondary resistance to 5-FC, on the other hand, is well documented to occur among both Candidaspp and Cryptococcus neoformans during monotherapy with this agent (292).

Resistance to 5-FC may develop from decreased uptake (loss of permease activity) or by loss of enzymatic activity required for the conversion of 5-FC to 5-FU (cytosine deaminase) and 5-fluorouridylic acid (FUMP pyrophosphorylase) (292,293). Of these possible mechanisms, the most important appear to be the loss of cytosine deaminase activity or the loss of UMP pyrophosphorylase activity. Uracil phosphoribosyltransferase, another enzyme in the primidine salvage pathway, is also important in the formation of FUMP and loss of its activity is sufficient to confer resistance to 5-FC (293).


Allylamine resistance has been infrequently reported despite clinical failures in close to 25% of patients treated with these agents (191). Laboratory-created resistant isolates have been found to carry extra copies of the ERG1 gene (294) or single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine (295). Subsequently, clinical isolates with amino acid substitutions within the ERG1 gene have also been reported (296,297). Sanglard et al. (267) have reported that the CDR1 multidrug efflux pump can use terbinafine as a substrate, thus the possibility of efflux-mediated resistance to allylamines exists.

Clinical Factors Contributing to Antifungal Resistance

Fungal infections may fail to respond to appropriate antifungal therapy, thereby demonstrating “clinical resistance,” despite the fact that the drug employed is active against the infecting organism. The interaction of the host, the drug, and the fungus is complex and clinical outcomes are influenced by a number of interactions (298).

During treatment of invasive fungal infections, the immune status of the host is the most important factor in determining outcomes. The presence of neutrophils, utilization of immunomodulating drugs, concomitant infections (e.g., HIV), surgical procedures, age, and nutritional status all may be more important than the ability, or lack thereof, of antifungal agents to inhibit or kill the infecting organism. Likewise, the site and severity of infection plays a critical role in the pharmacokinetic/pharmacodynamic interactions during antifungal therapy. The presence of a foreign body, such as catheter, prosthetic valve, or vascular graft material, may allow an otherwise susceptible organism to cause an infection that is recalcitrant to therapy with an otherwise active agent. Finally, an antifungal agent cannot act if the patient does not take it in the prescribed manner. Noncompliance is a major cause of apparent “resistance” to antifungal therapy and additionally may contribute to the development of resistant strains.

The absorption, distribution, and metabolism of an antifungal agent all contribute to the effectiveness of the drug at the site of infection. Insufficient (too low) dosing practices may influence both therapeutic efficacy and the potential for resistance development (299). The ability of an antifungal agent to exhibit fungicidal activity versus fungistatic activity may be especially important in severe infections, in infections where the organism burden is high, and in the neutropenic host (64,300). Drug–drug interactions may also affect the activity of an antifungal agent: drugs that are metabolized by the cytochrome P450 enzyme system, such as rifamycin, may dramatically decrease the achievable concentrations of azole antifungal agents such as itraconazole and voriconazole (301).

Fungal properties, aside from the expression of known resistance factors, may also impact the clinical success or failure of antifungal therapy. The different morphologic forms of fungi (e.g., blastospore, hypha, pseudohypha, conidia, chlamydospore) may all have different susceptibilities to various antifungal agents (302,303). Phenotypic switching in Candida has been shown to have a dramatic impact on the susceptibility of various species to polyenes and azoles (223,304). Finally, the rate of growth of a fungus and whether or not it is growing in a planktonic or biofilm form can determine whether it will require low or high concentrations of an antifungal to inhibit growth (305,306). Candida spp are increasingly resistant to antifungals when grown in a biofilm compared to those grown in solution (307).


The incidence of infection with invasive mycoses continues to rise with the increasing immunosuppressed patient population. The recently expanded antifungal armamentarium offers the potential for more effective and less toxic therapy and these agents offer distinct pharmacologic profiles and indications for use. An understanding of the differing spectrum of activity, pharmacokinetics/pharmacodynamics, and dosing regimens enables the clinician to provide patients the best chance of favorable outcomes.


The increasing number and diversity of invasive infections, expanding utilization of new and established antifungal agents, and recognition of antifungal resistance as an important clinical problem have contributed to the need for reproducible, clinically relevant antifungal susceptibility testing, especially for yeasts, but also for the filamentous fungi (298).

Rationale for Antifungal Susceptibility Testing

The central objective of all in vitro susceptibility testing is to help predict the likely impact of administration of the tested agent on the outcome of disease caused by the tested organism or similar organisms. As such, in vitro susceptibility tests of antifungal agents are performed for the same reasons as tests of antibacterial agents: (a) to provide a reliable estimate of the relative activities of antimicrobial agents, (b) to correlate with in vivo activity and predict the outcome of the therapy, (c) to provide a means by which to survey the development of resistance among a normally susceptible population of organisms, and (d) to predict the therapeutic potential of newly developed investigational agents. In the clinical microbiology/mycology laboratory, the focus of testing is on a specific isolate from an individual patient. In drug discovery, the focus of testing may be on the selection of the most potent of a series of compounds for further development. In antimicrobial resistance surveillance, the issue may be the tendency of resistance to emerge in initially susceptible isolates or species and to establish local, regional, or national patterns of resistance. In each of these settings, it is necessary to remember that outcome prediction is difficult and dependent on complex and dynamic biologic system and may substantially differ from results obtained in an artificial and well-defined matrix (antimicrobial susceptibility test). Decades of experience with antibacterial susceptibility testing confirms the limited degree of in vitro–in vivo correlation that can be achieved. The in vitro susceptibility of Enterococcus to trimethoprim-sulfamethoxazole and in vivo resistance to this combination is illustrative of this fact (308,309). In vitro susceptibility does not always predict successful therapy (298).

Development of Standardized Methods

At the present time, the state-of-the-art method for susceptibility testing of yeasts is comparable with that of bacteria (298). The CLSI Subcommittee on Antifungal Susceptibility Testing has developed and published approved methods for broth dilution testing of yeasts (310) and for disk diffusion testing of yeasts against fluconazole (311). These methods are reproducible, accurate, and available for use in clinical laboratories (312,313). Standardized methods have also been developed for broth dilution testing of filamentous fungi (314) but require further refinement and studies to establish the in vivo correlation with the in vitro data.

Standardized Broth Dilution Methods for Yeasts

In the United States, the National Committee for Clinical Laboratory Standards (NCCLS) (now CLSI) Subcommittee on Antifungal Susceptibility Testing was established in 1982 and focused on the key in vitro testing variables of inoculum preparation and size, medium composition, temperature and duration of incubation, and MIC end point determination in an effort to develop a standardized approach to antifungal susceptibility testing of yeasts (Candidaspp and Cryptococcus neoformans) using a broth dilution format (315319). As a result of these collaborative studies, consensus within the subcommittee was achieved on all of the variables, leading to the publication of a proposed broth macrodilution method, M27-P, in 1992 (320). This document was revised and published in 1995 as NCCLS document M27-T (tentative standard), which described the broth microdilution method and provided reference MIC ranges for two quality control (QC) strains for the available antifungal agents. In 1997, the subcommittee established interpretive MIC breakpoints for three antifungal agents (fluconazole, itraconazole, and 5-FC) (321) and the NCCLS-approved standard M27-A was published. Since then, the subcommittee has developed 24- and 48-hour reference QC MIC ranges for microdilution testing of both established (AmB, 5-FC, fluconazole, itraconazole, and ketoconazole) and newly introduced (voriconazole and posaconazole) agents (322). The results of these studies are included in the second edition of NCCLS document M27, M27-A2, published in 2002 (323). Since this time, the M27-A3 document was published in 2008 with the addition of caspofungin, micafungin, and anidulafungin susceptibility recommendations (324).

Adherence to the NCCLS M27-A3 method provides excellent intralaboratory and interlaboratory reproducibility and utilization of the recommended QC isolates will further ensure reliable test performance (324). The most recently published CLSI guidelines established revised breakpoints for fluconazole and voriconazole along with the echinocandins, which are species specific (Tables 6.4 and 6.5). These revised breakpoints recognize the wild-type distributions of Candida species against both azoles (fluconazole and voriconazole) and the echinocandins along with predicted pharmacokinetic and pharmacodynamic parameters of the drugs. These have generally resulted in much lower breakpoints for most species.

Standardized Disk Diffusion Methods for Yeasts and Nondermatophyte Filamentous Fungi

Disk diffusion testing has served as a simple, rapid, and cost-effective alternative to broth dilution testing of antibacterial agents for many years (311,325). Disk diffusion testing of antifungal agents has been slow to develop; however, early studies with fluconazole disks showed promise for testing Candida spp (326,327). Further development of this method has documented the precision and accuracy of the fluconazole disk diffusion test and has established QC zone diameter limits for both fluconazole and voriconazole when tested against Candida spp (328).

The NCCLS M44-A method uses Mueller-Hinton agar supplemented with 2% glucose and 0.5 µg/mL of methylene blue (311,312,329). The increased glucose and the methylene blue supplementation provides improved growth and sharper zones surrounding the fluconazole and voriconazole disks (312). This method employs an inoculum suspension adjusted to the turbidity of 0.5 McFarland Standard and 24-hour incubation at 35°C. The zone diameters surrounding the 25-µg fluconazole disks and the 1-µg voriconazole disks are read using reflected light and measured to the nearest whole millimeter at the point at which there is prominent reduction in growth. Pinpoint microcolonies at the zone edge or large colonies within a zone are ignored.

This method provides qualitative susceptibility results 24 hours sooner than the standard NCCLS M27-A2 MIC method for yeasts (330). The disk test results correlate well with reference MICs for both fluconazole and voriconazole and have allowed the establishment of zone interpretive criteria (breakpoints) for fluconazole and QC parameters for both fluconazole and voriconazole. The use of supplemented Mueller-Hinton agar in lieu of RPMI 1640 medium should make antifungal susceptibility testing available to a larger number of clinical laboratories at reduced cost. Since publication of the initial M44-A document, an update has been released (M44-A2) with zone diameter interpretive standards added for caspofungin, voriconazole, and posaconazole (311).

Similarly, the CLSI has developed a reference method for disk diffusion antifungal susceptibility testing of nondermatophyte filamentous fungi (325,331). These documents provide guidelines for testing the susceptibility of opportunistic molds to triazoles, AmB, and caspofungin and more recent publications have proposed QC and reference zone diameter limits for three strains selected as QC isolates (A. fumigatus ATCC MYA-3626, Paecilomyces variotii ATCC MYA-3630, and C. krusei ATCC 6258). These QC would assist in monitoring the performance of in vitro antifungal disk diffusion susceptibility testing by the CLSI M51-A and alternative methods (332). Additional studies are ongoing to further clarify testing condition with other echinocandins by this methodology (333).

Standardized Broth Dilution Methods for Filamentous Fungi

Serious infections due to filamentous fungi (molds), especially those due to Aspergillus spp, continue to increase and improving outcomes for these infections is an area of ongoing need. Given the increasing array of antifungal agents with systemic activity against the filamentous fungi, it is recognized that antifungal susceptibility testing of those opportunistic pathogens may be important in guiding the selection of antifungal agents for treatment of invasive disease. This is especially true for the newer triazoles (posaconazole and voriconazole) and echinocandins (caspofungin, micafungin, and anidulafungin) agents, all of which have varying degrees of activity against the opportunistic molds (171,334). Based on the achievements in standardizing in vitro susceptibility testing of yeasts, the CLSI antifungal subcommittee proceeded to develop a standardized method for the broth dilution testing of molds, NCCLS M38-A (335). The CLSI subcommittee used the M27-A2 microdilution method as a template for the development of the method for filamentous fungi. The approved method, M38-A, is applicable for testing Aspergillus spp, Fusarium spp, P. boydii, and Mucorales, and efforts remain ongoing to validate these methods for other agents (336). These guidelines were updated in 2008 with the publication of CLSI M38-A2 and provide MIC and minimum effective concentration (MEC) recommendations for the echinocandins, ciclopirox, griseofulvin, and terbinafine (314).

Progress and New Developments in Antifungal Susceptibility Testing

The availability of the M27 reference method for broth dilution testing of yeasts paved the way for the development of the reference method for filamentous fungi and was a necessary precursor to the disk diffusion methods. In addition, the M27 method has served as a cornerstone for the evaluation of new and improved methods for performing antifungal testing in the clinical laboratory and has facilitated the performance of large-scale national and international surveillance studies of the in vitro susceptibility of Candida spp and other fungi to both established and investigational antifungal agents (171,187,216,337339). These studies have allowed broad MIC distribution profiles for clinical isolates to be generated and are immensely helpful to clinicians in determining initial antifungal agents prior to isolate specific susceptibility results returning and for identifying isolates to be used in the characterization of resistance mechanisms (187,316,340). Finally, the standardization of antifungal susceptibility testing has made it possible to conduct nationwide studies of laboratory proficiency and to begin to establish the clinical relevance of antifungal susceptibility testing (341343).

New Test Development

The purpose of reference methods for antimicrobial susceptibility testing is to encourage standardization of the process and improve reproducibility among laboratories; however, reference methods may not be ideal for individual clinical laboratories. Other methods currently used include a microdilution format read spectrophotometrically or colorimetrically (344348).

In addition, novel breakpoint methods including culture on a porous aluminum oxide (PAO) support combined with microscopy (349) and flow cytometry (350,351) have been applied with varying degrees of success. The advent of whole-genome sequencing and rapidly decreasing costs for this method may in the future be the backbone behind susceptibility testing; however, the molecular changes responsible for phenotypic resistance in yeasts/molds remain incompletely defined and substantial work is needed in this area before genetic sequencing will replace in vitro susceptibility testing.

The utilization of colorimetric growth indicators has been applied to the microdilution method in an effort to provide improved ease and precision of MIC end point determination and possibly provide a more rapid means of testing clinically important fungi. Colorimetric determination of residual glucose (352,353), pH indicator dyes (354), and various tetrazolium salt methods (355,356) have all shown promise and are currently used in the Vitek 2 system employed in a large number of clinical laboratories (357359). The use of the oxidation reduction indicator alamarBlue (AccuMed International Inc, Chicago, IL) is one of the best studied and most widely used approaches (360362). In broth media, fungal growth causes the alamarBlue indicator to change from blue to pink. In an alamarBlue-containing system, the MIC for an antifungal agent is recorded as the first well to show a change from pink (growth) to purple or blue (growth inhibition). The alamarBlue indicator has been incorporated into a commercially available broth microdilution system, the Sensititre YeastOne colormetric antifungal plate (TREK Diagnostics Systems, Westlake, OH). The YeastOne system has been shown to perform comparably to the CLSI reference method (362364) and is approved by the U.S. Food and Drug Administration for in vitro susceptibility testing of yeasts.

The use of spectrophotometry has long been employed to measure the growth of microbes in a broth system. Spectrophotometric determination of fungal growth in the presence and absence of antifungal agents has been utilized to provide a more precise and objective means of MIC end point reading, especially for the azole class of antifungal agents (348,365,366).

In many clinical settings, determination of precise MICs using a full-range dilution series is not necessary. Testing of the clinical isolate against one or two antimicrobial drug concentrations that distinguish susceptible from resistant strains, so-called “breakpoint testing,” is often all that is necessary to allow the selection of optimal antifungal therapy (367). The agar-based methods for antifungal susceptibility testing include the previously mentioned disk diffusion method (NCCLS M44), the Etest stable agar gradient method (368373), and the semisolid agar dilution method (374). The Etest method (AB Biodisk, Solna, Sweden) has been widely employed as a means of producing an accurate, reproducible, and quantitative MIC result using an agar diffusion format (369). This method is based on the diffusion of a continuous concentration gradient of an antimicrobial agent from a plastic strip into an agar medium. When an Etest strip is placed on an agar plate inoculated with a test organism and incubated for 24 to 48 hours, an ellipse of growth inhibition occurs and the intersection of the ellipse with the numeric scale on the strip allows the reading of the MIC. This test method has been extensively studied for utilization in antibacterial testing and is comparable with the reference broth and agar dilution MIC methods. The Etest is also applicable to antifungal susceptibility testing. Numerous studies demonstrate the usefulness of Etest for determining the in vitro susceptibility of both yeasts and molds to a variety of antifungal agents including AmB, 5-FC, ketoconazole, itraconazole, voriconazole, posaconazole, and caspofungin. MICs determined by Etest generally agree quite well with those determined by the NCCLS reference method (312); however, this agreement may vary, depending on the antifungal agent tested, the choice of agar medium, and the organism species (372). The use of RPMI agar supplemented with 2% glucose works well for most organisms and antifungal agents, yet decreased agreement has been reported for fluconazole and itraconazole tested against Candida glabrata and Candida tropicalis (330,375). The use of Mueller-Hinton agar supplemented with glucose and methylene blue has been shown to improve the overall agreement of Etest MICs with the reference method when testing Candida glabrata against fluconazole and voriconazole (375). Of major importance is the fact that Etest is the most sensitive and reliable method for detecting decreased susceptibility to AmB among isolates of Candida spp and Cryptococcus neoformans (376379).

A semisolid agar dilution method was proposed by Provine and Hadley (374) to serve as a rapid, breakpoint screening test to detect isolates of Candida spp with decreased susceptibility to fluconazole. The method employs heart infusion broth with 0.5% agar and without glucose. The organism is inoculated into the system by stabbing the agar and the resulting conditions are considered to mimic more closely the growth conditions of infected tissue (374). This simple method provides good categorical agreement with the M27 reference method, although the number of isolates tested to date is limited.

Flow cytometric methods have been employed with good success in testing Candida spp against fluconazole and AmB (351,380). This approach uses a standard flow cytometer and fluorescent DNA binding dyes to detect fungal cell damage following exposure to an antifungal agent. The method produces results within 6 hours that agree very well with MICs determined by the M27 reference method.

Surveillance and New Drug Evaluation

One of the important offshoots of the standardization process has been the ability to conduct active surveillance of resistance to antifungal agents (187,216). Meaningful large-scale studies of antifungal susceptibility and resistance conducted over time would not be possible without a standardized microdilution method for performing the in vitro studies and several such studies have now been published. Furthermore, studies analyzing resistance trends to commonly utilized antifungal agents and comparative analyses of licensed and established antifungal agents have provided large amounts of useful data and have been greatly facilitated by a standardized microdilution method. As new antifungal agents are evaluated in clinical trials, in vitro susceptibility testing of clinical isolates utilizing a reference method will be essential and allow further establishment of in vitro/in vivo correlations.

Proficiency Testing

The participation of clinical laboratories in proficiency testing programs is considered an important step in ensuring quality and standardization in the performance of antimicrobial susceptibility testing (381). Prior to the publication of NCCLS document M27-A, little was known of the proficiency of clinical laboratories in performing antifungal susceptibility testing aside from those laboratories actively engaged in NCCLS-conducted studies. Following the publication in 1997 of the NCCLS M27-A reference method for testing yeasts, the College of American Pathologists initiated a proficiency-testing program for antifungal susceptibility testing, and following this, the number of participants has continued to increase and laboratory performance has steadily improved (382). This program provides important information regarding the performance of antifungal susceptibility testing in the United States and indicates a level of performance that is on par with that of antibacterial testing.

Global Standardization

Subsequent to the development of the NCCLS M27-A method for broth microdilution testing of yeasts, a similar method has been developed under the auspices of the Subcommittee on Antifungal Susceptibility Testing (AFST) of the European Committee on Antibiotic Susceptibility Testing (EUCAST) and periodically updated with technical notes on specific antifungal compounds (383388). This method is very similar to the NCCLS M27-A2 method and employs a higher inoculum (105 CFU/mL), RPMI 1640 medium supplemented with additional glucose (2%), and spectrophotometric readings of MIC end points following incubation at 35°C to 37°C for 24 hours (389). The efforts of the EUCAST-AFST subcommittee have stimulated a collaboration with the CLSI subcommittee and recent multicenter studies have documented good intralaboratory reproducibility of the EUCAST method (390) and good agreement between the EUCAST and CLSI microdilution methods (391). In addition, global surveillance programs such as the ARTEMIS global antifungal program for disk testing and MIC testing, the European Confederation of Medical Mycology survey of candidemia, and the SENTRY antifungal surveillance program promote the use of standardized disk and broth dilution MIC methods and provide useful and consistent antifungal susceptibility data from a broad network of hospitals and laboratories on an international scale.

Clinical Relevance of Antifungal Susceptibility Test Results

In order to be useful clinically, in vitro susceptibility testing of antimicrobial agents should reliably predict the in vivo response to therapy in human infections (Table 6.6). However, the in vitro susceptibility of an infecting organism to the antimicrobial agent is only one of several factors that may influence the likelihood that therapy for an infection will be successful (298). Factors related to the host immune response and/or the status of the current underlying disease, drug pharmacokinetics and pharmacodynamics, drug interactions, proper patient management, and factors related to the virulence of the infecting organism and its interaction with both the host and the antimicrobial agent administered all influence the outcome of treatment of an infectious episode (298,321). In order to appreciate the clinical value of antifungal susceptibility testing, one must understand that after more than 30 years of study, in vitro susceptibility testing can be said to predict the outcome of bacterial infections with an accuracy that has been summarized as the “90–60 rule” (298): infections due to susceptible isolates respond to therapy approximately 90% of the time, whereas infections due to resistant isolates respond approximately 60% of the time. There is now a considerable body of data indicating that standardized antifungal susceptibility testing (NCCLS M27-A3) for selected organism–drug combinations (most notably Candida spp and azole antifungal agents) provides results that have a predictive utility consistent with the 90–60 rule. The EUCAST breakpoints are significantly lower than those initially reported from the CLSI due in part to the fact that more cases of candidemia than OPC were used to calculate initial CLSI breakpoint values. Recent CLSI guidance more closely correlates with the EUCAST breakpoints. In addition, it is clear that breakpoints should ideally not cross the breakpoint for a wild-type population (i.e., the epidemiologic cutoff value or ECV) of a species. Theoretically, the ECV establishes the 95% breakpoint where isolates in a given species will not exhibit any resistance mechanisms and thus be more likely to be nonresponders in a clinical setting (392).Thus, the most recent CLSI breakpoint guidance establishes breakpoints for individual Candida species for both azoles (fluconazole and voriconazole) and the echinocandins (anidulafungin, caspofungin, and micafungin). In most cases, these breakpoints are significantly lower than those recommended in early documents and are based on predicted pharmacokinetic/pharmacodynamic parameters of the agents, wild-type distributions of the MICs and clinical/in vivo responses to the agents.

Of note in the new CLSI breakpoints are changes in C. glabrata susceptibility. Due to the wide distribution of wild-type C. glabrata MICs against both voriconazole and fluconazole and the potential for clinical resistance, breakpoints for fully susceptible C. glabrata have been eliminated for those agents. Fluconazole is reported as S-DD at an MIC of 32 or less and resistance at 64 or greater, whereas no susceptibility for voriconazole is recommended. Similarly, the breakpoints for full echinocandin susceptibility against C. glabrata for micafungin is now 0.06 µg/mL and for caspofungin/anidulafungin 0.12 µg/mL (due to differences in protein binding), with the recognition that isolates with higher MICs have a significant likelihood of harboring hot spot mutations in the target FSK1/2 genes.

Antifungal susceptibility testing is now increasingly and appropriately utilized as a routine adjunct to the treatment of fungal infections and guidelines for the utilization of antifungal testing, and other laboratory studies, have been developed (298). Selective application of antifungal susceptibility testing, coupled with broader identification of fungi to the species level, has proven useful especially in difficult-to-manage fungal infections. Future efforts will be dedicated to the further validation of interpretive breakpoints for established antifungal agents and developing them for newly introduced systemically active agents. In addition, procedures must be further refined for testing non-Candida yeasts (e.g., Trichosporon spp) and molds.


The determination of antifungal drug concentrations in serum, CSF, and other body fluids may provide clinicians with information that can be maximized to increase the probability of favorable patient outcomes. Numerous methods are available to determine drug levels in body fluids including microbiologic bioassay, gas–liquid chromatography, HPLC, UPLC/MS, fluorometry, spectrophotometry, thin-layer chromatography, and others. Physicochemical assay methods are more sensitive, specific, rapid, and less labor-intensive than bioassay methods. They are also capable of separating the parent compound from a biologically active metabolite, whereas bioassays are typically unable to do this (393395).

Bioassay methods are available for several of the systemically active antifungal agents. Bioassays have some advantages over physicochemical assays in that they do not require initial extraction steps or specialized instrumentation. In addition, bioassays evaluate the biologic activity of the antifungal agent, as well as any active metabolites, in the body fluid, whereas physiochemical determinations do not necessarily indicate biologically active drugs (343,393). The easiest and most practical bioassays to perform in the clinical laboratory are agar diffusion assays. AmB levels in body fluids are often determined by using an agar diffusion bioassay method. Briefly, molten agar is seeded with a standardized suspension of P. variotii (ATCC 36257), poured into 150-mL Petri dishes, and allowed to solidify on a level surface. Known concentrations of the antifungal agent to be tested, as well as the patient’s serum, are placed into wells cut from the agar and allowed to diffuse into the medium. The plates are incubated at 30°C for 24 to 48 hours, and the zones of inhibition around the reservoirs are measured to the nearest millimeter. A standard curve is plotted on semilogarithmic paper using the known concentrations of the drug and the corresponding zones of inhibition. Once the standard curve has been constructed, the drug levels in the patient’s body fluid may be determined by plotting the zone of inhibition on the standard curve. To ensure intralaboratory reproducibility, internal standards of known concentrations should be included with each assay.

AmB concentrations may be determined in the presence of 5-FC by substituting a 5-FC–resistant strain of Chrysosporium pruinosum (ATCC 36374) for P. variotii. The medium may also be supplemented with the 5-FC antagonist cytosine (10 µg/mL). These measures prevent the 5-FC from interfering with the determination of the AmB concentration.

5-FC concentrations in body fluids are determined in the same manner as those of AmB except that Saccharomyces cerevisiae (ATCC 36375) is substituted for P. variotii and yeast morphology agar is used instead of antibiotic medium 12. To assay for 5-FC in the presence of AmB, the latter is inactivated by heating the sample for 30 minutes at 90°C. Once the serum has been heated and the AmB inactivated, the sample may be assayed to determine the concentration of 5-FC present.

Microbiologic bioassays are also available to determine the concentrations of itraconazole, posaconazole, voriconazole, and the echinocandins in serum, plasma, or CSF (396). Importantly, the bioassay for itraconazole reflects the activity of both itraconazole and the hydroxylated metabolite, hydroxyitraconazole (393). The antifungal activity of these metabolites is unclear with a Candida kefyr isolate demonstrating twofold less activity of itraconazole than to hydroxyitraconazole (393), whereas other studies have shown a twofold difference in the opposite direction with itraconazole more active than hydroxyitraconazole (differences of only two dilutions fall well within the range of normal replicate variability) (397,398).

These findings have obvious implications in the interpretation of bioassay results for itraconazole and will be highly discrepant when compared with itraconazole concentrations determined by physiochemical methods unless the physiochemical determination of hydroxyitraconazole is also taken into account such as with UPLC/MS (396). In contrast, metabolism of voriconazole results in inactive metabolites and an excellent correlation between bioassay and HPLC determinations of voriconazole and posaconazole in serum has been reported (394,399).


In response to the challenge of invasive mycoses and the development of resistance by several of the non-albicans Candida species, the number of systemically active agents has increased over the past several years. As a result of this challenge, greater understanding of the mechanism of action of these agents as well as the capability of fungal pathogens to demonstrate resistance is needed. Antifungal susceptibility testing has undergone continued standardized refinement; it is generally considered to play a significant role in the clinical management of invasive mycoses. Guidelines for the role of antifungal susceptibility testing have been clearly codified and structured. Although significant progress has been made in these areas, additional efforts are needed to optimize testing methods for newly developed antifungal compounds and for testing pathogens other than Candida. Ongoing national and international investigations and collaborations targeted to address these several issues will serve to refine and improve the management of invasive fungal infections.


The authors thank Drs. Michael Pfaller, Daniel J. Diekema, and Michael G. Rinaldi for the content of this chapter’s previous edition, which has been included in part within this update.


  1.  Thompson GR 3rd, Cadena J, Patterson TF. Overview of antifungal agents. Clin Chest Med 2009;30(2):203–215, v.

  2.  Ellis D. Amphotericin B: spectrum and resistance. J Antimicrob Chemother 2002;49(Suppl 1):7–10.

  3.  Amphotericin B [package insert]. Big Flats, NY: X-Gen Pharmaceuticals; 2009.

  4.  Ben-Ami R, Lewis RE, Kontoyiannis DP. Immunocompromised hosts: immunopharmacology of modern antifungals. Clin Infect Dis 2008;47(2):226–235.

  5.  Bellocchio S, Gaziano R, Bozza S, et al. Liposomal amphotericin B activates antifungal resistance with reduced toxicity by diverting toll-like receptor signalling from TLR-2 to TLR-4. J Antimicrob Chemother 2005;55(2):214–222.

  6.  Steinbach WJ, Benjamin DK Jr, Kontoyiannis DP, et al. Infections due to Aspergillus terreus: a multicenter retrospective analysis of 83 cases. Clin Infect Dis 2004;39(2):192–198.

  7.  Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007;20(4):695–704.

  8.  Meletiadis J, Meis JF, Mouton JW, et al. In vitro activities of new and conventional antifungal agents against clinical Scedosporium isolates. Antimicrob Agents Chemother 2002;46(1):62–68.

  9.  Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet 2002;359(9312):1135–1144.

 10.  Blum G, Perkhofer S, Haas H, et al. Potential basis for amphotericin B resistance in Aspergillus terreusAntimicrob Agents Chemother 2008;52(4):1553–1555.

 11.  White MH, Anaissie EJ, Kusne S, et al. Amphotericin B colloidal dispersion vs. amphotericin B as therapy for invasive aspergillosis. Clin Infect Dis 1997;24(4):635–642.

 12.  Mistro S, Maciel Ide M, de Menezes RG, et al. Does lipid emulsion reduce amphotericin B nephrotoxicity? A systematic review and meta-analysis. Clin Infect Dis 2012;54(12):1774–1777.

 13.  Collette N, van der Auwera P, Lopez AP, et al. Tissue concentrations and bioactivity of amphotericin B in cancer patients treated with amphotericin B-deoxycholate. Antimicrob Agents Chemother 1989;33(3):362–368.

 14.  Perfect JR, Dismukes WE, Dromer F, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2010;50(3):291–322.

 15.  Stevens DA, Shatsky SA. Intrathecal amphotericin in the management of coccidioidal meningitis. Semin Respir Infect 2001;16(4):263–269.

 16.  Bekersky I, Fielding RM, Dressler DE, et al. Pharmacokinetics, excretion, and mass balance of liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate in humans. Antimicrob Agents Chemother 2002;46(3):828–833.

 17.  Wiederhold NP, Tam VH, Chi J, et al. Pharmacodynamic activity of amphotericin B deoxycholate is associated with peak plasma concentrations in a neutropenic murine model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother 2006;50(2):469–473.

 18.  Andes D. In vivo pharmacodynamics of antifungal drugs in treatment of candidiasis. Antimicrob Agents Chemother 2003;47(4):1179–1186.

 19.  Saag MS, Graybill RJ, Larsen RA, et al. Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin Infect Dis 2000;30(4):710–718.

 20.  Dodds Ashley ES, Lewis R, Lewis JS, et al. Pharmacology of systemic antifungal agents. Clin Infect Dis 2006;43(Suppl 1):S28–S39.

 21.  Pappas PG, Rex JH, Sobel JD, et al. Guidelines for treatment of candidiasis. Clin Infect Dis 2004;38(2):161–189.

 22.  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. Candidemia Study Group and the National Institute. N Engl J Med 1994;331(20):1325–1330.

 23.  Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 2002;347(6):408–415.

 24.  Freifeld AG, Bow EJ, Sepkowitz KA, et al. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011;52(4):427–431.

 25.  Johnson PC, Wheat LJ, Cloud GA, et al. Safety and efficacy of liposomal amphotericin B compared with conventional amphotericin B for induction therapy of histoplasmosis in patients with AIDS. Ann Intern Med 2002;137(2):105–109.

 26.  Sun HY, Alexander BD, Lortholary O, et al. Lipid formulations of amphotericin B significantly improve outcome in solid organ transplant recipients with central nervous system cryptococcosis. Clin Infect Dis 2009;49(11):1721–1728.

 27.  Walsh TJ, Anaissie EJ, Denning DW, et al. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008;46(3):327–360.

 28.  Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis. Clin Infect Dis 2008;47(4):503–509.

 29.  Borro JM, Sole A, de la Torre M, et al. Efficiency and safety of inhaled amphotericin B lipid complex (abelcet) in the prophylaxis of invasive fungal infections following lung transplantation. Transplant Proc 2008;40(9):3090–3093.

 30.  Slobbe L, Boersma E, Rijnders BJ. Tolerability of prophylactic aerosolized liposomal amphotericin-B and impact on pulmonary function: data from a randomized placebo-controlled trial. Pulm Pharmacol Ther 2008;21(6):855–859.

 31.  Rijnders BJ, Cornelissen JJ, Slobbe L, et al. Aerosolized liposomal amphotericin B for the prevention of invasive pulmonary aspergillosis during prolonged neutropenia: a randomized, placebo-controlled trial. Clin Infect Dis 2008;46(9):1401–1408.

 32.  Mora-Duarte J, Betts R, Rotstein C, et al. Comparison of caspofungin and amphotericin B for invasive candidiasis. N Engl J Med 2002;347(25):2020–2029.

 33.  Saliba F, Dupont B. Renal impairment and amphotericin B formulations in patients with invasive fungal infections. Med Mycol 2008;46(2):97–112.

 34.  Kleinberg M. What is the current and future status of conventional amphotericin B? Int J Antimicrob Agents 2006;27(Suppl 1):12–16.

 35.  Johnson EM, Szekely A, Warnock DW. In-vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J Antimicrob Chemother 1998;42(6):741–745.

 36.  Hope WW, Billaud EM, Lestner J, et al. Therapeutic drug monitoring for triazoles. Curr Opin Infect Dis 2008;21(6):580–586.

 37.  Shimokawa O, Nakayama H. Increased sensitivity of Candida albicans cells accumulating 14 alpha-methylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents. Antimicrob Agents Chemother 1992;36(8):1626–1629.

 38.  Fluconazole [package insert]. New York: Pfizer Laboratories; 2004.

 39.  Itraconazole [package insert]. (Sempera) product monograph. Neuss, Germany: Janssen-Cilag GmbH; 2003.

 40.  Posaconazole [package insert]. Kenilworth, NJ: Schering Corporation; 2006.

 41.  Voriconazole [package insert]. New York: Pfizer Inc; 2002.

 42.  Gubbins PO, Heldenbrand S. Clinically relevant drug interactions of current antifungal agents. Mycoses 2010;53(2):95–113.

 43.  Kramer MR, Marshall SE, Denning DW, et al. Cyclosporine and itraconazole interaction in heart and lung transplant recipients. Ann Intern Med 1990;113(4):327–329.

 44.  Varis T, Kaukonen KM, Kivisto KT, et al. Plasma concentrations and effects of oral methylprednisolone are considerably increased by itraconazole. Clin Pharmacol Ther 1998;64(4):363–368.

 45.  Tucker RM, Denning DW, Hanson LH, et al. Interaction of azoles with rifampin, phenytoin, and carbamazepine: in vitro and clinical observations. Clin Infect Dis 1992;14(1):165–174.

 46.  Kivisto KT, Lamberg TS, Kantola T, et al. Plasma buspirone concentrations are greatly increased by erythromycin and itraconazole. Clin Pharmacol Ther 1997;62(3):348–354.

 47.  Engels FK, Ten Tije AJ, Baker SD, et al. Effect of cytochrome P450 3A4 inhibition on the pharmacokinetics of docetaxel. Clin Pharmacol Ther 2004;75(5):448–454.

 48.  Grub S, Bryson H, Goggin T, et al. The interaction of saquinavir (soft gelatin capsule) with ketoconazole, erythromycin and rifampicin: comparison of the effect in healthy volunteers and in HIV-infected patients. Eur J Clin Pharmacol 2001;57(2):115–121.

 49.  Jeng MR, Feusner J. Itraconazole-enhanced vincristine neurotoxicity in a child with acute lymphoblastic leukemia. Pediatr Hematol Oncol 2001;18(2):137–142.

 50.  Itraconazole [package insert]. (Sempera) product monograph. Neuss, Germany: Janssen-Cilag GmbH; 2003.

 51.  Kaukonen KM, Olkkola KT, Neuvonen PJ. Itraconazole increases plasma concentrations of quinidine. Clin Pharmacol Ther 1997;62(5):510–517.

 52.  Lefebvre RA, Van Peer A, Woestenborghs R. Influence of itraconazole on the pharmacokinetics and electrocardiographic effects of astemizole. Br J Clin Pharmacol 1997;43(3):319–322.

 53.  Honig PK, Wortham DC, Hull R, et al. Itraconazole affects single-dose terfenadine pharmacokinetics and cardiac repolarization pharmacodynamics. J Clin Pharmacol 1993;33(12):1201–1206.

 54.  Posaconazole [package insert]. Kenilworth, NJ: Schering Corporation; 2006.

 55.  Voriconazole (V-fend) [package insert]. Summary of Product Characteristics SAahemou.

 56.  DeMuria D, Forrest A, Rich J, et al. Pharmacokinetics and bioavailability of fluconazole in patients with AIDS. Antimicrob Agents Chemother 1993;37(10):2187–2192.

 57.  Blum RA, D’Andrea DT, Florentino BM, et al. Increased gastric pH and the bioavailability of fluconazole and ketoconazole. Ann Intern Med 1991;1149(9):755.

 58.  Arndt CA, Walsh TJ, McCully CL, et al. Fluconazole penetration into cerebrospinal fluid: implications for treating fungal infections of the central nervous system. J Infect Dis 1988;157(1):178–180.

 59.  Savani DV, Perfect JR, Cobo LM, et al. Penetration of new azole compounds into the eye and efficacy in experimental Candida endophthalmitis. Antimicrob Agents Chemother 1987;31(1):6–10.

 60.  Andes D, van Ogtrop M. Characterization and quantitation of the pharmacodynamics of fluconazole in a neutropenic murine disseminated candidiasis infection model. Antimicrob Agents Chemother 1999;43(9):2116–2120.

 61.  Klepser ME, Wolfe EJ, Pfaller MA. Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B against Cryptococcus neoformansJ Antimicrob Chemother 1998;41(3):397–401.

 62.  Louie A, Drusano GL, Banerjee P, et al. Pharmacodynamics of fluconazole in a murine model of systemic candidiasis. Antimicrob Agents Chemother 1998;42(5):1105–1109.

 63.  Orozco AS, Higginbotham LM, Hitchcock CA, et al. Mechanism of fluconazole resistance in Candida kruseiAntimicrob Agents Chemother 1998;42(10):2645–2649.

 64.  Baddley JW, Patel M, Bhavnani SM, et al. Association of fluconazole pharmacodynamics with mortality in patients with candidemia. Antimicrob Agents Chemother 2008;52(9):3022–3028.

 65.  Lortholary O, Denning DW, Dupont B. Endemic mycoses: a treatment update. J Antimicrob Chemother 1999;43(3):321–331.

 66.  Zonios DI, Bennett JE. Update on azole antifungals. Semin Respir Crit Care 2008;29(2):198–210.

 67.  Wheat J, Marichal P, Vanden Bossche H, et al. Hypothesis on the mechanism of resistance to fluconazole in Histoplasma capsulatumAntimicrob Agents Chemother 1997;41(2):410–414.

 68.  Perea S, Patterson TF. Antifungal resistance in pathogenic fungi. Clin Infect Dis 2002;35(9):1073–1080.

 69.  Pappas PG, Kauffman CA, Andes D, et al. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 2009;48(5):503–535.

 70.  Galgiani JN, Ampel NM, Blair JE, et al. Coccidioidomycosis. Clin Infect Dis 2005;41(9):1217–1223.

 71.  Hamza OJ, Matee MI, Bruggemann RJ, et al. Single-dose fluconazole versus standard 2-week therapy for oropharyngeal candidiasis in HIV-infected patients: a randomized, double-blind, double-dummy trial. Clin Infect Dis 2008;47(10):1270–1276.

 72.  Saag MS, Cloud GA, Graybill JR, et al. A comparison of itraconazole versus fluconazole as maintenance therapy for AIDS-associated cryptococcal meningitis. National Institute of Allergy and Infectious Diseases Mycoses Study Group. Clin Infect Dis 1999;28(2):291–296.

 73.  Longley N, Muzoora C, Taseera K, et al. Dose response effect of high-dose fluconazole for HIV-associated cryptococcal meningitis in southwestern Uganda. Clin Infect Dis 2008;47(12):1556–1561.

 74.  Johnson RH, Einstein HE. Coccidioidal meningitis. Clin Infect Dis 2006;42(1):103–107.

 75.  Wheat LJ, Freifeld AG, Kleiman MB, et al. Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the Infectious Diseases Society of America. Clin Infect Dis 2007;45(7):807–825.

 76.  Kauffman CA, Bustamante B, Chapman SW, et al. Clinical practice guidelines for the management of sporotrichosis: 2007 update by the Infectious Diseases Society of America. Clin Infect Dis 2007;45(10):1255–1265.

 77.  Slavin MA, Osborne B, Adams R, et al. Efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation—a prospective, randomized, double-blind study. J Infect Dis 1995;171(6):1545–1552.

 78.  Schuster MG, Edwards JE Jr, Sobel JD, et al. Empirical fluconazole versus placebo for intensive care unit patients: a randomized trial. Ann Intern Med 2008;149(2):83–90.

 79.  Stevens DA, Diaz M, Negroni R, et al. Safety evaluation of chronic fluconazole therapy. Fluconazole Pan-American Study Group. Chemotherapy 1997;43(5):371–377.

 80.  Dodds Ashley ES, Lewis R, Lewis JS, et al. Pharmacology of systemic antifungal agents. Clin Infect Dis 2006;43(Suppl 1):S28–S39.

 81.  Miyama T, Takanaga H, Matsuo H, et al. P-glycoprotein-mediated transport of itraconazole across the blood-brain barrier. Antimicrob Agents Chemother 1998;42(7):1738–1744.

 82.  Sheehan DJ, Hitchcock CA, Sibley CM. Current and emerging azole antifungal agents. Clin Microbiol Rev 1999;12(1):40–79.

 83.  Groll AH, Kolve H. Antifungal agents: in vitro susceptibility testing, pharmacodynamics, and prospects for combination therapy. Eur J Clin Microbiol Infect Dis 2004;23(4):256–270.

 84.  Manavathu EK, Cutright JL, Chandrasekar PH. Organism-dependent fungicidal activities of azoles. Antimicrob Agents Chemother 1998;42(11):3018–3021.

 85.  Verweij PE, Mellado E, Melchers WJ. Multiple-triazole-resistant aspergillosis. N Engl J Med 2007;356(14):1481–1483.

 86.  Verweij PE, Voss A, Meis JF. Resistance of Aspergillus fumigatus to itraconazole. Scand J Infect Dis 1998;30(6):642–643.

 87.  Barone JA, Moskovitz BL, Guarnieri J, et al. Enhanced bioavailability of itraconazole in hydroxypropyl-beta-cyclodextrin solution versus capsules in healthy volunteers. Antimicrob Agents Chemother 1998;42(7):1862–1865.

 88.  Van de Velde VJ, Van Peer AP, Heykants JJ, et al. Effect of food on the pharmacokinetics of a new hydroxypropyl-beta-cyclodextrin formulation of itraconazole. Pharmacology 1996;16(3):424–428.

 89.  Stevens DA. Itraconazole in cyclodextrin solution. Pharmacotherapy 1999;19(5):603–611.

 90.  Como JA, Dismukes WE. Oral azole drugs as systemic antifungal therapy. N Engl J Med 1994;330(4):263–272.

 91.  Warnock DW, Turner A, Burke J. Comparison of high performance liquid chromatographic and microbiological methods for determination of itraconazole. J Antimicrob Chemother 1988;21(1):93–100.

 92.  Limper AH, Knox KS, Sarosi GA, et al. An official American Thoracic Society statement: treatment of fungal infections in adult pulmonary and critical care patients. Am J Respir Crit Care Med 2011;183(1):96–128.

 93.  Chapman SW, Dismukes WE, Proia LA, et al. Clinical practice guidelines for the management of blastomycosis: 2008 update by the Infectious Diseases Society of America. Clin Infect Dis 2008;46(12):1801–1812.

 94.  Restrepo A, Benard G, de Castro CC, et al. Pulmonary paracoccidioidomycosis. Semin Respir Crit Care Med 2008;29(2):182–197.

 95.  Galgiani JN, Catanzaro A, Cloud GA, et al. Comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis. A randomized, double-blind trial. Mycoses Study Group. Ann Intern Med 2000;133(9):676–686.

 96.  Revankar SG. Phaeohyphomycosis. Infect Dis Clin North Am 2006;20(3):609–620.

 97.  De Beule K, Van Gestel J. Pharmacology of itraconazole. Drugs 2001;61(Suppl 1):27–37.

 98.  Sharkey PK, Rinaldi MG, Dunn JF, et al. High-dose itraconazole in the treatment of severe mycoses. Antimicrob Agents Chemother 1991;35(4):707–713.

 99.  Tucker RM, Haq Y, Denning DW, et al. Adverse events associated with itraconazole in 189 patients on chronic therapy. J Antimicrob Chemother 1990;26(4):561–566.

100.  Glasmacher A, Hahn C, Molitor E, et al. Itraconazole through concentrations in antifungal prophylaxis with six different dosing regimens using hydroxypropyl-beta-cyclodextrin oral solution or coated-pellet capsules. Mycoses 1999;42(11–12):591–600.

101.  Ahmad SR, Singer SJ, Leissa BG. Congestive heart failure associated with itraconazole. Lancet 2001;357(9270):1766–1767.

102.  Manavathu EK, Cutright JL, Loebenberg D, et al. A comparative study of the in vitro susceptibilities of clinical and laboratory-selected resistant isolates of Aspergillus spp. to amphotericin B, itraconazole, voriconazole and posaconazole (SCH 56592). J Antimicrob Chemother 2000;46(2):229–234.

103.  Nagappan V, Deresinski S. Reviews of anti-infective agents: posaconazole: a broad-spectrum triazole antifungal agent. Clin Infect Dis 2007;45(12):1610–1617.

104.  Courtney R, Wexler D, Radwanski E, et al. Effect of food on the relative bioavailability of two oral formulations of posaconazole in healthy adults. Br J Clin Pharmacol 2004;57(2):218–222.

105.  Ezzet F, Wexler D, Courtney R, et al. Oral bioavailability of posaconazole in fasted healthy subjects: comparison between three regimens and basis for clinical dosage recommendations. Clin Pharmacokinet 2005;44(2):211–220.

106.  Jain R, Pottinger P. The effect of gastric acid on the absorption of posaconazole. Clin Infect Dis 2008;46(10):1627; author reply 1627–1628.

107.  Krishna G, Ma L, Malavade D, et al. Effect of gastric pH, dosing regimen and prandial state, food and meal timing relative to dose, and gastrointestinal motility on absorption and pharmacokinetics of the antifungal posaconazole. In: 18th European Congress of Clinical Microbiology and Infectious Diseases; March. Barcelona, Spain. 2008:P1264.

108.  Courtney R, Pai S, Laughlin M, et al. Pharmacokinetics, safety, and tolerability of oral posaconazole administered in single and multiple doses in healthy adults. Antimicrob Agents Chemother 2003;47(9):2788–2795.

109.  Sansone-Parsons A, Krishna G, Calzetta A, et al. Effect of a nutritional supplement on posaconazole pharmacokinetics following oral administration to healthy volunteers. Antimicrob Agents Chemother 2006;50(5):1881–1883.

110.  Meletiadis J, Chanock S, Walsh TJ. Human pharmacogenomic variations and their implications for antifungal efficacy. Clin Microbiol Rev 2006;19(4):763–787.

111.  Cornely OA, Maertens J, Winston DJ, et al. Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007;356(4):348–359.

112.  Ullmann AJ, Lipton JH, Vesole DH, et al. Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007;356(4):335–347.

113.  Walsh TJ, Raad I, Patterson TF, et al. Treatment of invasive aspergillosis with posaconazole in patients who are refractory to or intolerant of conventional therapy: an externally controlled trial. Clin Infect Dis 2007;44(1):2–12.

114.  Lehrnbecher T, Attarbaschi A, Duerken M, et al. Posaconazole salvage treatment in paediatric patients: a multicentre survey. Eur J Clin Microbiol Infect Dis 2010;29(8):1043–1045.

115.  Courtney R, Sansone A, Smith W, et al. Posaconazole pharmacokinetics, safety, and tolerability in subjects with varying degrees of chronic renal disease. J Clin Pharmacol 2005;45(2):185–192.

116.  Raad II, Graybill JR, Bustamante AB, et al. Safety of long-term oral posaconazole use in the treatment of refractory invasive fungal infections. Clin Infect Dis 2006;42(12):1726–1734.

117.  Johnson LB, Kauffman CA. Voriconazole: a new triazole antifungal agent. Clin Infect Dis 2003;36(5):630–637.

118.  Purkins L, Wood N, Kleinermans D, et al. Effect of food on the pharmacokinetics of multiple-dose oral voriconazole. Br J Clin Pharmacol 2003;56(Suppl 1):17–23.

119.  Thompson GR 3rd, Lewis JS 2nd. Pharmacology and clinical use of voriconazole. Expert Opin Drug Metab Toxicol 2010;6(1):83–94.

120.  Purkins L, Wood N, Ghahramani P, et al. Pharmacokinetics and safety of voriconazole following intravenous- to oral-dose escalation regimens. Antimicrob Agents Chemother 2002;46(8):2546–2553.

121.  Hariprasad SM, Mieler WF, Holz ER, et al. Determination of vitreous, aqueous, and plasma concentration of orally administered voriconazole in humans. Arch Ophthalmol 2004;122(1):42–47.

122.  Nierenberg NE, Thompson GR, Lewis JS, et al. Voriconazole use and pharmacokinetics in combination with interferon-gamma for refractory cryptococcal meningitis in a patient receiving low-dose ritonavir. Med Mycol 2010;48(3):532–536.

123.  Ikeda Y, Umemura K, Kondo K, et al. Pharmacokinetics of voriconazole and cytochrome P450 2C19 genetic status. Clin Pharmacol Ther 2004;75(6):587–588.

124.  Klepser ME, Malone D, Lewis RE, et al. Evaluation of voriconazole pharmacodynamics using time-kill methodology. Antimicrob Agents Chemother 2000;44(7):1917–1920.

125.  Andes D, Marchillo K, Stamstad T, et al. In vivo pharmacokinetics and pharmacodynamics of a new triazole, voriconazole, in a murine candidiasis model. Antimicrob Agents Chemother 2003;47(10):3165–3169.

126.  Krishnan S, Manavathu EK, Chandrasekar PH. A comparative study of fungicidal activities of voriconazole and amphotericin B against hyphae of Aspergillus fumigatusJ Antimicrob Chemother 2005;55(6):914–920.

127.  Kirkpatrick WR, McAtee RK, Fothergill AW, et al. Efficacy of voriconazole in a guinea pig model of disseminated invasive aspergillosis. Antimicrob Agents Chemother 2000;44(10):2865–2868.

128.  Nguyen MH, Yu CY. Voriconazole against fluconazole-susceptible and resistant Candida isolates: in-vitro efficacy compared with that of itraconazole and ketoconazole. J Antimicrob Chemother 1998;42(2):253–256.

129.  Pfaller MA, Messer SA, Hollis RJ, et al. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob Agents Chemother 2002;46(6):1723–1727.

130.  Sutton DA, Sanche SE, Revankar SG, et al. In vitro amphotericin B resistance in clinical isolates of Aspergillus terreus, with a head-to-head comparison to voriconazole. J Clin Microbiol 1999;37(7):2343–2345.

131.  Cuenca-Estrella M, Ruiz-Diez B, Martinez-Suarez JV, et al. Comparative in-vitro activity of voriconazole (UK-109,496) and six other antifungal agents against clinical isolates of Scedosporium prolificans and Scedosporium apiospermumJ Antimicrob Chemother1999;43(1):149–151.

132.  Lamaris GA, Ben-Ami R, Lewis RE, et al. Increased virulence of Zygomycetes organisms following exposure to voriconazole: a study involving fly and murine models of zygomycosis. J Infect Dis 2009;199(9):1399–1406.

133.  Denning DW, Ribaud P, Milpied N, et al. Efficacy and safety of voriconazole in the treatment of acute invasive aspergillosis. Clin Infect Dis 2002;34(5):536–571.

134.  Perfect JR, Marr KA, Walsh TJ, et al. Voriconazole treatment for less-common, emerging, or refractory fungal infections. Clin Infect Dis 2003;36(9):1122–1131.

135.  Kullberg BJ, Sobel JD, Ruhnke M, et al. Voriconazole versus a regimen of amphotericin B followed by fluconazole for candidaemia in non-neutropenic patients: a randomised non-inferiority trial. Lancet 2005;366(9495):1435–1442.

136.  Ally R, Schurmann D, Kreisel W, et al. A randomized, double-blind, double-dummy, multicenter trial of voriconazole and fluconazole in the treatment of esophageal candidiasis in immunocompromised patients. Clin Infect Dis 2001;33(9):1447–1454.

137.  Walsh TJ, Pappas P, Winston DJ, et al. Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N Engl J Med 2002;346(4):225–234.

138.  Kinoshita J, Iwata N, Ohba M, et al. Mechanism of voriconazole-induced transient visual disturbance: reversible dysfunction of retinal ON-bipolar cells in monkeys. Invest Ophthalmol Vis Sci 2011;52(8):5058–5063.

139.  Scherpbier HJ, Hilhorst MI, Kuijpers TW. Liver failure in a child receiving highly active antiretroviral therapy and voriconazole. Clin Infect Dis 2003;37(6):828–830.

140.  Pascual A, Calandra T, Bolay S, et al. Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes. Clin Infect Dis 2008;46(2):201–211.

141.  Lewis RE. What is the “therapeutic range” for voriconazole? Clin Infect Dis 2008;46(2):212–214.

142.  Zonios DI, Gea-Banacloche J, Childs R, et al. Hallucinations during voriconazole therapy. Clin Infect Dis 2008;47(1):e7–e10.

143.  Chapman SW, Bradsher RW Jr, Campbell GD Jr, et al. Practice guidelines for the management of patients with blastomycosis. Infectious Diseases Society of America. Clin Infect Dis 2000;30(4):679–683.

144.  Kauffman CA, Hajjeh R, Chapman SW. Practice guidelines for the management of patients with sporotrichosis. For the Mycoses Study Group. Infectious Diseases Society of America. Clin Infect Dis 2000;30(4):684–687.

145.  Goodwin ML, Drew RH. Antifungal serum concentration monitoring: an update. J Antimicrob Chemother 2008;61(1):17–25.

146.  Onishi J, Meinz M, Thompson J, et al. Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 2000;44(2):368–377.

147.  Cappelletty D, Eiselstein-McKitrick K. The echinocandins. Pharmacotherapy 2007;27(3):369–388.

148.  Abe F, Ueyama J, Kawasumi N, et al. Role of plasma proteins in pharmacokinetics of micafungin, an antifungal antibiotic, in analbuminemic rats. Antimicrob Agents Chemother 2008;52(9):3454–3456.

149.  Andes D, Diekema DJ, Pfaller MA, et al. In vivo comparison of the pharmacodynamic targets for echinocandin drugs against Candida species. Antimicrob Agents Chemother 2010;54(6):2497–2506.

150.  Deresinski SC, Stevens DA. Caspofungin. Clin Infect Dis 2003;36(11):1445–1457.

151.  Lat A, Thompson GR 3rd, Rinaldi MG, et al. Micafungin concentrations from brain tissue and pancreatic pseudocyst fluid. Antimicrob Agents Chemother 2010;54(2):943–944.

152.  Kethireddy S, Andes D. CNS pharmacokinetics of antifungal agents. Expert Opin Drug Metab Toxicol 2007;3(4):573–581.

153.  Keating GM, Jarvis B. Caspofungin. Drugs 2001;61(8):1121–1129; discussion 30–31.

154.  Chandrasekar PH, Sobel JD. Micafungin: a new echinocandin. Clin Infect Dis 2006;42(8):1171–1178.

155.  Andes D, Diekema DJ, Pfaller MA, et al. In vivo pharmacodynamic characterization of anidulafungin in a neutropenic murine candidiasis model. Antimicrob Agents Chemother 2008;52(2):539–550.

156.  Ernst EJ, Klepser ME, Pfaller MA. Postantifungal effects of echinocandin, azole, and polyene antifungal agents against Candida albicans and Cryptococcus neoformansAntimicrob Agents Chemother 2000;44(4):1108–1111.

157.  Bowman JC, Hicks PS, Kurtz MB, et al. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 2002;46(9):3001–3012.

158.  Petraitiene R, Petraitis V, Groll AH, et al. Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship to galactomannan antigenemia. Antimicrob Agents Chemother 2002;46(1):12–23.

159.  Petraitis V, Petraitiene R, Groll AH, et al. Comparative antifungal activities and plasma pharmacokinetics of micafungin (FK463) against disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits. Antimicrob Agents Chemother2002;46(6):1857–1869.

160.  Ryan DM, Lupinacci RJ, Kartsonis NA. Efficacy and safety of caspofungin in obese patients. Med Mycol 2011;49(7):748–754.

161.  Hall RG, Swancutt MA, Gumbo T. Fractal geometry and the pharmacometrics of micafungin in overweight, obese, and extremely obese people. Antimicrob Agents Chemother 2011;55(11):5107–5112.

162.  Wiederhold NP. Paradoxical echinocandin activity: a limited in vitro phenomenon? Med Mycol 2009;47(Suppl 1):S369–S375.

163.  Wiederhold NP. Attenuation of echinocandin activity at elevated concentrations: a review of the paradoxical effect. Curr Opin Infect Dis 2007;20(6):574–578.

164.  Pappas PG, Rotstein CM, Betts RF, et al. Micafungin versus caspofungin for treatment of candidemia and other forms of invasive candidiasis. Clin Infect Dis 2007;45(7):883–893.

165.  Riddell J, Comer GM, Kauffman CA. Treatment of endogenous fungal endophthalmitis: focus on new antifungal agents. Clin Infect Dis 2011;52(5):648–653.

166.  Pfaller MA, Diekema DJ, Ostrosky-Zeichner L, et al. Correlation of MIC with outcome for Candida species tested against caspofungin, anidulafungin, and micafungin: analysis and proposal for interpretive MIC breakpoints. J Clin Microbiol 2008;46(8):2620–2629.

167.  Lamaris GA, Lewis RE, Chamilos G, et al. Caspofungin-mediated beta-glucan unmasking and enhancement of human polymorphonuclear neutrophil activity against Aspergillus and non-Aspergillus hyphae. J Infect Dis 2008;198(2):186–192.

168.  Nakai T, Uno J, Otomo K, et al. In vitro activity of FK463, a novel lipopeptide antifungal agent, against a variety of clinically important molds. Chemotherapy 2002;48(2):78–81.

169.  Espinel-Ingroff A. Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK-0991 (L-743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts. J Clin Microbiol 1998;36(10):2950–2956.

170.  Ramani R, Chaturvedi V. Antifungal susceptibility profiles of Coccidioides immitis and Coccidioides posadasii from endemic and non-endemic areas. Mycopathologica 2007;163(6):315–319.

171.  Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J Clin Microbiol 2003;41(8):3623–3626.

172.  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):324–326.

173.  Pfaller MA, Boyken L, Hollis RJ, et al. In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance. J Clin Microbiol 2008;46(1):150–156.

174.  Andes DR, Safdar N, Baddley JW, et al. Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials. Clin Infect Dis 2012;54(8):1110–1122.

175.  Kuse ER, Chetchotisakd P, da Cunha CA, et al. Micafungin versus liposomal amphotericin B for candidaemia and invasive candidosis: a phase III randomised double-blind trial. Lancet 2007;369(9572):1519–1527.

176.  Reboli AC, Rotstein C, Pappas PG, et al. Anidulafungin versus fluconazole for invasive candidiasis. N Engl J Med 2007;356(24):2472–2482.

177.  Cornely OA, Lasso M, Betts R, et al. Caspofungin for the treatment of less common forms of invasive candidiasis. J Antimicrob Chemother 2007;60(2):363–369.

178.  Baddley JW, Benjamin DK Jr, Patel M, et al. Candida infective endocarditis. Eur J Clin Microbiol Infect Dis 2008;27(7):519–529.

179.  Heinz WJ, Einsele H. Caspofungin for treatment of invasive Aspergillus infections. Mycoses 2008;51(Suppl 1): 47–57.

180.  Marr KA, Hachem R, Papanicolaou G, et al. Retrospective study of the hepatic safety profile of patients concomitantly treated with caspofungin and cyclosporin A. Transplant Infect Dis 2004;6(3):110–116.

181.  Sable CA, Nguyen BY, Chodakewitz JA, et al. Safety and tolerability of caspofungin acetate in the treatment of fungal infections. Transpl Infect Dis 2002;4(1):25–30.

182.  Polak A, Scholer HJ. Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 1975;21(3–4):113–130.

183.  Vermes A, Guchelaar HJ, Dankert J. Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J Antimicrob Chemother 2000;46(2):171–179.

184.  Lewis RE, Klepser ME, Pfaller MA. In vitro pharmacodynamic characteristics of flucytosine determined by time-kill methods. Diagn Microbiol Infect Dis 2000;36(2):101–105.

185.  Andes D, van Ogtrop M. In vivo characterization of the pharmacodynamics of flucytosine in a neutropenic murine disseminated candidiasis model. Antimicrob Agents Chemother 2000;44(4):938–942.

186.  Brouwer AE, Rajanuwong A, Chierakul W, et al. Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial. Lancet 2004; 363(9423):1764–1767.

187.  Brandt ME, Pfaller MA, Hajjeh RA, et al. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates in the United States: 1992 to 1994 and 1996 to 1998. Antimicrob Agents Chemother 2001;45(11):3065–3069.

188.  Pfaller MA, Messer SA, Boyken L, et al. In vitro activities of 5-fluorocytosine against 8,803 clinical isolates of Candida spp.: global assessment of primary resistance using National Committee for Clinical Laboratory Standards susceptibility testing methods. Antimicrob Agents Chemother2002;46(11):3518–3521.

189.  Vermes A, van Der Sijs H, Guchelaar HJ. Flucytosine: correlation between toxicity and pharmacokinetic parameters. Chemotherapy 2000;46(2):86–94.

190.  Stamm AM, Diasio RB, Dismukes WE, et al. Toxicity of amphotericin B plus flucytosine in 194 patients with cryptococcal meningitis. Am J Med 1987;83(2):236–242.

191.  Ghannoum MA, Rice LB. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12(4):501–517.

192.  Krishnan-Natesan S. Terbinafine: a pharmacological and clinical review. Expert Opin Pharmacother 2009;10(16):2723–2733.

193.  Jessup CJ, Ryder NS, Ghannoum MA. An evaluation of the in vitro activity of terbinafine. Med Mycol 2000;38(2):155–159.

194.  McGinnis MR, Pasarell L. In vitro evaluation of terbinafine and itraconazole against dematiaceous fungi. Med Mycol 1998;36(4):243–246.

195.  McGinnis MR, Nordoff NG, Ryder NS, et al. In vitro comparison of terbinafine and itraconazole against Penicillium marneffei. Antimicrob Agents Chemother 2000; 44(5):1407–1408.

196.  Ryder NS. Activity of terbinafine against serious fungal pathogens. Mycoses 1999;42(Suppl 2):115–119.

197.  Gupta AK, Shear NH. Terbinafine: an update. J Am Acad Dermatol 1997;37(6):979–988.

198.  Faergemann J, Anderson C, Hersle K, et al. Double-blind, parallel-group comparison of terbinafine and griseofulvin in the treatment of toenail onychomycosis. J Am Acad Dermatol 1995;32(5 Pt 1):750–753.

199.  Hofmann H, Brautigam M, Weidinger G, et al. Treatment of toenail onychomycosis. A randomized, double-blind study with terbinafine and griseofulvin. LAGOS II Study Group. Arch Dermatol 1995;131(8):919–922.

200.  Ghannoum MA, Elewski B. Successful treatment of fluconazole-resistant oropharyngeal candidiasis by a combination of fluconazole and terbinafine. Clin Diagn Lab Immunol 1999;6(6):921–923.

201.  Esterre P, Inzan CK, Ramarcel ER, et al. Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study. Br J Dermatol 1996;134(Suppl 46): 33–36; discussion 40.

202.  Terbinafine [package insert]. East Hanover, NJ: Novartis Pharmaceuticals Corporation; 2012.

203.  Hall M, Monka C, Krupp P, et al. Safety of oral terbinafine: results of a postmarketing surveillance study in 25,884 patients. Arch Dermatol 1997;133(10):1213–1219.

204.  Ornstein DL, Ely P. Reversible agranulocytosis associated with oral terbinafine for onychomycosis. J Am Acad Dermatol 1998;39(6):1023–1024.

205.  De Carli L, Larizza L. Griseofulvin. Mutat Res 1988; 195(2):91–126.

206.  Balfour JA, Faulds D. Terbinafine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in superficial mycoses. Drugs 1992; 43(2):259–284.

207.  Fleece D, Gaughan JP, Aronoff SC. Griseofulvin versus terbinafine in the treatment of tinea capitis: a meta-analysis of randomized, clinical trials. Pediatrics 2004; 114(5):1312–1315.

208.  Gonzalez U, Seaton T, Bergus G, et al. Systemic antifungal therapy for tinea capitis in children. Cochrane Database Syst Rev 2007;(4):CD004685.

209.  Elewski BE, Caceres HW, DeLeon L, et al. Terbinafine hydrochloride oral granules versus oral griseofulvin suspension in children with tinea capitis: results of two randomized, investigator-blinded, multicenter, international, controlled trials. J Am Acad Dermatol 2008; 59(1):41–45.

210.  Lipozencic J, Skerlev M, Orofino-Costa R, et al. A randomized, double-blind, parallel-group, duration-finding study of oral terbinafine and open-label, high-dose griseofulvin in children with tinea capitis due to Microsporum species. Br J Dermatol 2002;146(5):816–823.

211.  Griseofulvin [package insert]. Lincoln, NE: Novartis Pharmaceuticals Corporation; 2009.

212.  Darouiche RO. Oropharyngeal and esophageal candidiasis in immunocompromised patients: treatment issues. Clin Infect Dis 1998;26(2):259–272; quiz 73–74.

213.  Kaplan JE, Benson C, Holmes KH, et al. Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep 2009;58(RR-4):1–207; quiz CE1–CE4.

214.  Boucher HW, Talbot GH, Bradley JS, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009;48(1):1–12.

215.  Johnson EM. Issues in antifungal susceptibility testing. J Antimicrob Chemother 2008;61(Suppl 1):i13–i18.

216.  Cleveland AA, Farley MM, Harrison LH, et al. Changes in incidence and antifungal drug resistance in candidemia: results from population-based laboratory surveillance in Atlanta and Baltimore, 2008-2011. Clin Infect Dis 2012;55(10):1352–1361.

217.  Pfaller MA, Castanheira M, Lockhart SR, et al. Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrataJ Clin Microbiol 2012; 50(4):1199–1203.

218.  White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998;11(2):382–402.

219.  Sanglard D, Odds FC. Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2002;2(2):73–85.

220.  Cannon RD, Lamping E, Holmes AR, et al. Efflux-mediated antifungal drug resistance. Clin Microbiol Rev 2009;22(2):291–321, Table of Contents.

221.  White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997;41(7):1482–1487.

222.  Franz R, Kelly SL, Lamb DC, et al. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrob Agents Chemother 1998;42(12):3065–3072.

223.  Favel A, Michel-Nguyen A, Peyron F, et al. Colony morphology switching of Candida lusitaniae and acquisition of multidrug resistance during treatment of a renal infection in a newborn: case report and review of the literature. Diagnostic Microbiol Infect Dis 2003; 47(1):331–339.

224.  Kanafani ZA, Perfect JR. Antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis 2008;46(1):46–48.

225.  Rex JH, Cooper CR Jr, Merz WG, et al. Detection of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrob Agents Chemother 1995;39(4): 906–909.

226.  Blignaut E, Molepo J, Pujol C, et al. Clade-related amphotericin B resistance among South African Candida albicans isolates. Diagn Microbiol Infect Dis 2005; 53(1):29–231.

227.  Pfaller MA, Diekema DJ, Gibbs DL, et al. Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to 2005. J Clin Microbiol 2008;46(2):515–521.

228.  Pfaller MA, Diekema DJ, Messer SA, et al. In vitro activities of voriconazole, posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood. J Clin Microbiol 2003;41(1):78–83.

229.  Escribano P, Pelaez T, Recio S, et al. Characterization of clinical strains of Aspergillus terreus complex: molecular identification and antifungal susceptibility to azoles and amphotericin B. Clin Microbiol Infect 2012;18(2): E24–E26.

230.  Lass-Florl C, Alastruey-Izquierdo A, Cuenca-Estrella M, et al. In vitro activities of various antifungal drugs against Aspergillus terreus: global assessment using the methodology of the European Committee on Antimicrobial Susceptibility testing. Antimicrob Agents Chemother 2009;53(2):794–795.

231.  Kelly SL, Lamb DC, Taylor M, et al. Resistance to amphotericin B associated with defective sterol delta 8—>7 isomerase in a Cryptococcus neoformans strain from an AIDS patient. FEMS Microbiol Lett 1994;122(1–2):39–42.

232.  Witt MD, Lewis RJ, Larsen RA, et al. Identification of patients with acute AIDS-associated cryptococcal meningitis who can be effectively treated with fluconazole: the role of antifungal susceptibility testing. Clin Infect Dis 1996;22(2):322–328.

233.  Athar MA, Winner HI. The development of resistance by Candida species to polyene antibiotics in vitro. J Med Microbiol 1971;4(4):505–517.

234.  Dick JD, Rosengard BR, Merz WG, et al. Fatal disseminated candidiasis due to amphotericin-B-resistant Candida guilliermondiiAnn Intern Med 1985;102(1):67–68.

235.  Dick JD, Merz WG, Saral R. Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob Agents Chemother 1980;18(1):158–163.

236.  Powderly WG, Kobayashi GS, Herzig GP, et al. Amphotericin B-resistant yeast infection in severely immunocompromised patients. Am J Med 1988;84(5):826–832.

237.  Safe LM, Safe SH, Subden RE, et al. Sterol content and polyene antibiotic resistance in isolates of Candida kruseiCandida parakrusei, and Candida tropicalisCan J Microbiol 1977;23(4):398–401.

238.  Yoon SA, Vazquez JA, Steffan PE, et al. High-frequency, in vitro reversible switching of Candida lusitaniae clinical isolates from amphotericin B susceptibility to resistance. Antimicrob Agents Chemother 1999;43(4):836–845.

239.  Hitchcock CA, Barrett-Bee KJ, Russell NJ. The lipid composition and permeability to azole of an azole- and polyene-resistant mutant of Candida albicansJ Med Vet Mycol 1987.

240.  Subden RE, Safe L, Morris DC, et al Eburicol, lichesterol, ergosterol, and obtusifoliol from polyene antibiotic-resistant mutants of Candida albicansCan J Microbiol 1977;23(6):751–754.

241.  Sokol-Anderson M, Sligh JE Jr, Elberg S, et al. Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B. Antimicrob Agents Chemother 1988;32(5):702–705.

242.  Nolte FS, Parkinson T, Falconer DJ, et al. Isolation and characterization of fluconazole- and amphotericin B-resistant Candida albicans from blood of two patients with leukemia. Antimicrob Agents Chemother 1997;41(1): 196–199.

243.  Fryberg M, Oehlschlager AC, Unrau AM. Sterol biosynthesis in antibiotic-resistant yeast: nystatin. Arch Biochem Biophys 1974;160(1):83–89.

244.  Gale EF, Johnson AM, Kerridge D, et al. Factors affecting the changes in amphotericin sensitivity of Candida albicans during growth. J Gen Microbiol 1975;87(1): 20–36.

245.  Morio F, Pagniez F, Lacroix C, et al. Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence. J Antimicrob Chemother 2012;67(9):2131–2138.

246.  Akins RA. An update on antifungal targets and mechanisms of resistance in Candida albicansMed Mycol 2005;43(4):285–318.

247.  Pfaller MA, Diekema DJ. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of CandidaClin Microbiol Infect 2004;10(Suppl 1):11–23.

248.  Lockhart SR, Iqbal N, Cleveland AA, et al. Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011. J Clin Microbiol 2012;50(11):3435–3442.

249.  Fukuoka T, Johnston DA, Winslow CA, et al. Genetic basis for differential activities of fluconazole and voriconazole against Candida kruseiAntimicrob Agents Chemother 2003;47(4):1213–1219.

250.  Sipsas NV, Lewis RE, Tarrand J, et al. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009;115(20):4745–4752.

251.  Ghannoum MA, Okogbule-Wonodi I, Bhat N, et al. Antifungal activity of voriconazole (UK-109,496), fluconazole and amphotericin B against hematogenous Candida krusei infection in neutropenic guinea pig model. J Chemother 1999;11(1):34–39.

252.  Cuenca-Estrella M, Diaz-Guerra TM, Mellado E, et al. Comparative in vitro activity of voriconazole and itraconazole against fluconazole-susceptible and fluconazole-resistant clinical isolates of Candida species from Spain. Eur J Clin Microbiol Infect Dis 1999;18(6): 432–435.

253.  Ostrosky-Zeichner L, Oude Lashof AM, Kullberg BJ, et al. Voriconazole salvage treatment of invasive candidiasis. Eur J Clin Microbiol Infect Dis 2003;22(11): 651–655.

254.  Pfaller MA, Diekema DJ, Jones RN, et al. Trends in antifungal susceptibility of Candida spp. isolated from pediatric and adult patients with bloodstream infections: SENTRY Antimicrobial Surveillance Program, 1997 to 2000. J Clin Microbiol 2002;40(3):852–856.

255.  Majithiya J, Sharp A, Parmar A, et al. Efficacy of isavuconazole, voriconazole and fluconazole in temporarily neutropenic murine models of disseminated Candida tropicalis and Candida kruseiJ Antimicrob Chemother 2009;63(1):161–166.

256.  Pfaller MA, Messer SA, Boyken L, et al. In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp. and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program. Diagn Microbiol Infect Dis2004;48(3):201–205.

257.  Spreghini E, Maida CM, Tomassetti S, et al. Posaconazole against Candida glabrata isolates with various susceptibilities to fluconazole. Antimicrob Agents Chemother 2008;52(6):1929–1933.

258.  Espinel-Ingroff A, Aller AI, Canton E, et al. Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole. Antimicrob Agents Chemother 2012;56(11):5898–5906.

259.  Thompson GR 3rd, Wiederhold NP, Fothergill AW, et al. Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformansAntimicrob Agents Chemother 2009;53(1):309–311.

260.  Casadevall A, Spitzer ED, Webb D, et al. Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrob Agents Chemother 1993;37(6):1383–1386.

261.  Baddley JW, Marr KA, Andes DR, et al. Patterns of susceptibility of Aspergillus isolates recovered from patients enrolled in the Transplant-Associated Infection Surveillance Network. J Clin Microbiol 2009;47(10):3271–3275.

262.  Verweij PE, Kema GH, Zwaan B, et al. Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatusPest Manag Sci 2012.

263.  Snelders E, Camps SM, Karawajczyk A, et al. Triazole fungicides can induce cross-resistance to medical triazoles in Aspergillus fumigatusPLoS One 2012;7(3):e31801.

264.  Mosquera J, Denning DW. Azole cross-resistance in Aspergillus fumigatusAntimicrob Agents Chemother 2002; 46(2):556–557.

265.  White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicansAntimicrob Agents Chemother 1997;47(7): 1488–1494.

266.  Sanglard D, Ischer F, Calabrese D, et al. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob Agents Chemother 1999; 43(11):2753–2765.

267.  Sanglard D, Ischer F, Monod M, et al. Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 1997;143(Pt 2):405–416.

268.  Sanglard D, Kuchler K, Ischer F, et al. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 1995; 39(11):2378–2386.

269.  MacCallum DM, Coste A, Ischer F, et al. Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection. Antimicrob Agents Chemother 2010; 54(4):1476–1483.

270.  Lopez-Ribot JL, McAtee RK, Lee LN, et al. Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother1998;42(11):2932–2937.

271.  Lopez-Ribot JL, McAtee RK, Perea S, et al. Multiple resistant phenotypes of Candida albicans coexist during episodes of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 1999;43(7):1621–1630.

272.  Redding S, Smith J, Farinacci G, et al. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis 1994;18(2):240–242.

273.  Snelders E, van der Lee HA, Kuijpers J, et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med 2008;5(11):e219.

274.  Camps SM, Dutilh BE, Arendrup MC, et al. Discovery of a hapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing. PLoS One 2012;7(11):e50034.

275.  Joseph-Horne T, Hollomon D, Loeffler RS, et al. Cross-resistance to polyene and azole drugs in Cryptococcus neoformansAntimicrob Agents Chemother 1995;39(7): 1526–1529.

276.  Lamb DC, Corran A, Baldwin BC, et al. Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients. FEBS Lett 1995;368(2):326–330.

277.  Sionov E, Chang YC, Garraffo HM, et al. Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob Agents Chemother 2009;53(7):2804–2815.

278.  Pfaller MA, Messer SA, Boyken L, et al. Caspofungin activity against clinical isolates of fluconazole-resistant CandidaJ Clin Microbiol 2003;41(12):5729–5731.

279.  Pfaller MA, Diekema DJ, Messer SA, et al. In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates. Antimicrob Agents Chemother 2003;47(3):1068–1071.

280.  Kurtz MB, Abruzzo G, Flattery A, et al. Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies. Infect Immun 1996;64(8):3244–3251.

281.  Douglas CM, D’Ippolito JA, Shei GJ, et al. Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 1997;41(11):2471–2479.

282.  Thompson GR 3rd, Wiederhold NP, Vallor AC, et al. Development of caspofungin resistance following prolonged therapy for invasive candidiasis secondary to Candida glabrata infection. Antimicrob Agents Chemother 2008;52(10):3783–3785.

283.  Arendrup MC, Perkhofer S, Howard SJ, et al. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother 2008;52(10):3504–3511.

284.  Kahn JN, Garcia-Effron G, Hsu MJ, et al. Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase. Antimicrob Agents Chemother 2007;51(5):1876–1878.

285.  Park S, Kelly R, Kahn JN, et al. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob Agents Chemother 2005;49(8): 3264–3273.

286.  Katiyar S, Pfaller M, Edlind T. Candida albicans and Candida glabrata clinical isolates exhibiting reduced echinocandin susceptibility. Antimicrob Agents Chemother 2006;50(8):2892–2894.

287.  Beauvais A, Bruneau JM, Mol PC, et al. Glucan synthase complex of Aspergillus fumigatusJ Bacteriol 2001;183(7): 2273–2279.

288.  Gardiner RE, Souteropoulos P, Park S, et al. Characterization of Aspergillus fumigatus mutants with reduced susceptibility to caspofungin. Med Mycol 2005; 43(Suppl 1):S299–S305.

289.  Romano J, Nimrod G, Ben-Tal N, et al. Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence. Microbiology 2006;152(Pt 7): 1919–1928.

290.  Stiller RL, Bennett JE, Scholer HJ, et al. Susceptibility to 5-fluorocytosine and prevalence of serotype in 402 Candida albicans isolates from the United States. Antimicrob Agents Chemother 1982;22(3):482–487.

291.  Defever KS, Whelan WL, Rogers AL, et al. Candida albicans resistance to 5-fluorocytosine: frequency of partially resistant strains among clinical isolates. Antimicrob Agents Chemother 1982;22(5):810–815.

292.  Whelan WL. The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformansCerit Rev Microbiol 1987;15(1):45–56.

293.  Whelan WL, Kerridge D. Decreased activity of UMP pyrophosphorylase associated with resistance to 5-fluorocytosine in Candida albicansAntimicrob Agents Chemother 1984;26(4):570–574.

294.  Liu W, May GS, Lionakis MS, et al. Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine: genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatusAntimicrob Agents Chemother2004;48(7):2490–2496.

295.  Leber R, Fuchsbichler S, Klobucnikova V, et al. Molecular mechanism of terbinafine resistance in Saccharomyces cerevisiaeAntimicrob Agents Chemother 2003;47(12): 3890–3900.

296.  Osborne CS, Leitner I, Hofbauer B, et al. Biological, biochemical, and molecular characterization of a new clinical Trichophyton rubrum isolate resistant to terbinafine. Antimicrob Agents Chemother

297.  Osborne CS, Leitner I, Favre B, et al. Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine. Antimicrob Agents Chemother 2005;49(7):2840–2844.

298.  Rex JH, Pfaller MA. Has antifungal susceptibility testing come of age? Clin Infect Dis 2002;35(8):982–989.

299.  Pittrow L, Penk A. Special pharmacokinetics of fluconazole in septic, obese and burn patients. Mycoses 1999; 42(Suppl 2):87–90.

300.  Pfaller MA, Sheehan DJ, Rex JH. Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clin Microbiol Rev 2004;17(2):268–280.

301.  Lat A, Thompson GR 3rd. Update on the optimal use of voriconazole for invasive fungal infections. Infect Drug Resist 2011;4:43–53.

302.  Fernandez-Torres B, Inza I, Guarro J. Comparison of in vitro antifungal susceptibilities of conidia and hyphae of dermatophytes with thick-wall macroconidia. Antimicrob Agents Chemother 2003;47(10):3371–3372.

303.  Perkhofer S, Jost D, Dierich MP, et al. Susceptibility testing of anidulafungin and voriconazole alone and in combination against conidia and hyphae of Aspergillus spp. under hypoxic conditions. Antimicrob Agents Chemother 2008;52(5):1873–1875.

304.  Vargas K, Messer SA, Pfaller M, et al. Elevated phenotypic switching and drug resistance of Candida albicans from human immunodeficiency virus-positive individuals prior to first thrush episode. J Clin Microbiol 2000; 38(10):3595–3607.

305.  Al-Fattani MA, Douglas LJ. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J Med Microbiol 2006;55(Pt 8):999–1008.

306.  Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother 2000;46(3): 397–403.

307.  Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 2001;183(13):5385–5395.

308.  Chenoweth CE, Robinson KA, Schaberg DR. Efficacy of ampicillin versus trimethoprim-sulfamethoxazole in a mouse model of lethal enterococcal peritonitis. Antimicrob Agents Chemother 1990;34(9):1800–1802.

309.  Grayson ML, Thauvin-Eliopoulos C, Eliopoulos GM, et al. Failure of trimethoprim-sulfamethoxazole therapy in experimental enterococcal endocarditis. Antimicrob Agents Chemother 1990;34(9):1792–1794.

310.  Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. Wayne, PA: Clinical and Laboratory Standards Institute, 2008. CLSI document M27-A3.

311.  Clinical and Laboratory Standards Institute. Method for antifungal disk diffusion susceptibility testing of yeasts; approved standard. Wayne, PA: Clinical and Laboratory Standards Institute, 2008. CLSI document M44-A2.

312.  Barry AL, Pfaller MA, Rennie RP, et al. Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods. Antimicrob Agents Chemother 2002;46(6):1781–1784.

313.  Pfaller MA, Diekema DJ, Sheehan DJ. Interpretive breakpoints for fluconazole and Candida revisited: a blueprint for the future of antifungal susceptibility testing. Clin Microbiol Rev 2006;19(2):435–447.

314.  Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Wayne, PA: Clinical and Laboratory Standards Institute, 2008. CLSI document M38-A2.

315.  Fromtling RA, Galgiani JN, Pfaller MA, et al. Multicenter evaluation of a broth macrodilution antifungal susceptibility test for yeasts. Antimicrob Agents Chemother 1993;37(1):39–45

316.  Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 2012; 50(9):2846–2856.

317.  Pfaller MA, Castanheira M, Messer SA, et al. Echinocandin and triazole antifungal susceptibility profiles for Candida spp., Cryptococcus neoformans, and Aspergillus fumigatus: application of new CLSI clinical breakpoints and epidemiologic cutoff values to characterize resistance in the SENTRY Antimicrobial Surveillance Program (2009). Diagn Microbiol Infect Dis 2011;69(1):45–50.

318.  Pfaller MA, Rinaldi MG, Galgiani JN, et al. Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrob Agents Chemother 1990;34(9): 1648–1654.

319.  Pfaller MA, Burmeister L, Bartlett MS, et al. Multicenter evaluation of four methods of yeast inoculum preparation. J Clin Microbiol 1988;26(8):1437–1441.

320.  National Committee for Clinical Laboratory Standards. Reference method for broth dilution susceptibility testing of yeasts: proposed standard. Villanova, PA: National Committee for Clinical Laboratory Standards, 1992. NCCLS document M27-P.

321.  Rex JH, Pfaller MA, Galgiani JN, et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards. Clin Infect Dis 1997; 24(2):235–247.

322.  Barry AL, Pfaller MA, Brown SD, et al. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J Clin Microbiol 2000;38(9):3457–3459.

323.  National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Wayne, PA: National Committee for Clinical Laboratory Standards, 2002. NCCLS document M27-A2.

324.  Hata K, Kimura J, Miki H, et al. Efficacy of ER-30346, a novel oral triazole antifungal agent, in experimental models of aspergillosis, candidiasis, and cryptococcosis. Antimicrob Agents Chemother 1996;40(10):2243–2247.

325.  Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard, 2nd ed. CLSI document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute, 2008.

326.  Liebowitz LD, Ashbee HR, Evans EG, et al. A two year global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion. Diagn Microbiol Infect Dis 2001;40(1–2):27–33.

327.  Bille J, Glauser MP. Evaluation of the susceptibility of pathogenic Candida species to fluconazole. Fluconazole Global Susceptibility Study Group. Eur J Clin Microbiol Infect Dis 1997;16(12):924–928.

328.  Pfaller MA, Diekema DJ, Rinaldi MG, et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study: a 6.5-year analysis of susceptibilities of Candida and other yeast species to fluconazole and voriconazole by standardized disk diffusion testing. J Clin Microbiol 2005;43(12):5848–5859.

329.  Barry A, Bille J, Brown S, et al. Quality control limits for fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol 2003;41(7):3410–3412.

330.  Pfaller MA, Diekema DJ, Messer SA, et al. Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by Broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001. J Clin Microbiol 2003;41(4):1440–1446.

331.  Clinical and Laboratory Standards Institute. Method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline, 2nd ed. CLSI document M44-A2. Wayne, PA: Clinical and Laboratory Standards Institute, 2009.

332.  Espinel-Ingroff A, Canton E, Fothergill A, et al. Quality control guidelines for amphotericin B, Itraconazole, posaconazole, and voriconazole disk diffusion susceptibility tests with nonsupplemented Mueller-Hinton Agar (CLSI M51-A document) for nondermatophyte Filamentous Fungi. J Clin Microbiol 2011;49(7):2568–2571.

333.  Martos AI, Martin-Mazuelos E, Romero A, et al. Evaluation of disk diffusion method compared to broth microdilution for antifungal susceptibility testing of 3 echinocandins against Aspergillus spp. Diagn Microbiol Infect Dis 2012;73(1):53–56.

334.  Espinel-Ingroff A, Boyle K, Sheehan DJ. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 2001;150(3):101–115.

335.  National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Wayne, PA: National Committee for Clinical Laboratory Standards, 2002. NCCLS document M38-A.

336.  Ghannoum MA, Arthington-Skaggs B, Chaturvedi V, et al. Interlaboratory study of quality control isolates for a broth microdilution method (modified CLSI M38-A) for testing susceptibilities of dermatophytes to antifungals. J Clin Microbiol 2006;44(12):4353–4356.

337.  Chen YC, Chang SC, Luh KT, et al. Stable susceptibility of Candida blood isolates to fluconazole despite increasing use during the past 10 years. J Antimicrob Chemother 2003;52(1):71–77.

338.  Pfaller M, Neofytos D, Diekema D, et al. Epidemiology and outcomes of candidemia in 3648 patients: data from the Prospective Antifungal Therapy (PATH Alliance®) registry, 2004-2008. Diagn Microbiol Infect Dis 2012;74(4):323–331.

339.  Lepak A, Castanheira M, Diekema D, et al. Optimizing echinocandin dosing and susceptibility breakpoint determination via in vivo pharmacodynamic evaluation against Candida glabrata with and without fks mutations. Antimicrob Agents Chemother 2012;56(11):5875–5882.

340.  Canton E, Peman J, Quindos G, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosisCandida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother2011;55(12):5590–5596.

341.  Ramani R, Chaturvedi V. Proficiency testing program for clinical laboratories performing antifungal susceptibility testing of pathogenic yeast species. J Clin Microbiol 2003;41(3):1143–1146.

342.  Ranque S, Lachaud L, Gari-Toussaint M, et al. Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method. J Clin Microbiol 2012;50(7):2305–2309.

343.  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):3475–3482.

344.  Canton E, Peman J, Hervas D, et al. Comparison of three statistical methods for establishing tentative wild-type population and epidemiological cutoff values for echinocandins, amphotericin B, flucytosine, and six Candida species as determined by the colorimetric Sensititre YeastOne method. J Clin Microbiol 2012;50(12):3921–3926.

345.  Pfaller MA, Chaturvedi V, Diekema DJ, et al. Comparison of the Sensititre YeastOne colorimetric antifungal panel with CLSI microdilution for antifungal susceptibility testing of the echinocandins against Candida spp., using new clinical breakpoints and epidemiological cutoff values. Diagn Microbiol Infect Dis 2012;73(4):365–368.

346.  Radetsky M, Wheeler RC, Roe MH, et al. Microtiter broth dilution method for yeast susceptibility testing with validation by clinical outcome. J Clin Microbiol 1986;24(4):600–606.

347.  Nett JE, Cain MT, Crawford K, et al. Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay. J Clin Microbiol 2011;49(4):1426–1433.

348.  Cuenca-Estrella M, Moore CB, Barchiesi F, et al. Multicenter evaluation of the reproducibility of the proposed antifungal susceptibility testing method for fermentative yeasts of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Clin Microbiol Infect 2003;9(6):467–474.

349.  Ingham CJ, Boonstra S, Levels S, et al. Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide. PLoS One 2012;7(3):e33818.

350.  Rudensky B, Broide E, Berko N, et al. Direct fluconazole susceptibility testing of positive Candida blood cultures by flow cytometry. Mycoses 2008;51(3):200–204.

351.  Ramani R, Chaturvedi V. Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test. Antimicrob Agents Chemother 2000;44(10):2752–2758.

352.  Li RK, Elie CM, Clayton GE, et al. Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination. J Clin Microbiol 2000;38(6):2334–2338.

353.  Riesselman MH, Hazen KC, Cutler JE. Determination of antifungal MICs by a rapid susceptibility assay. J Clin Microbiol 2000;38(1):333–340.

354.  Fournier C, Gaspar A, Boillot F, et al. Evaluation of a broth microdilution antifungal susceptibility test with a pH indicator: comparison with the broth macrodilution procedures. J Antimicrob Chemother 1995;35(3): 373–380.

355.  Hawser SP, Norris H, Jessup CJ, et al. Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents. J Clin Microbiol 1998;36(5):1450–1452.

356.  Jahn B, Martin E, Stueben A, et al. Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. J Clin Microbiol 1995;33(3):661–667.

357.  Borghi E, Iatta R, Sciota R, et al. Comparative evaluation of the Vitek 2 yeast susceptibility test and CLSI broth microdilution reference method for testing antifungal susceptibility of invasive fungal isolates in Italy: the GISIA3 study. J Clin Microbiol 2010;48(9):3153–3157.

358.  Peterson JF, Pfaller MA, Diekema DJ, et al. Multicenter comparison of the Vitek 2 antifungal susceptibility test with the CLSI broth microdilution reference method for testing caspofungin, micafungin, and posaconazole against Candida spp. J Clin Microbiol 2011;49(5): 1765–1771.

359.  Pfaller MA, Diekema DJ, Procop GW, et al. Multicenter comparison of the VITEK 2 yeast susceptibility test with the CLSI broth microdilution reference method for testing fluconazole against Candida spp. J Clin Microbiol 2007;45(3):796–802.

360.  Jahn B, Stuben A, Bhakdi S. Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests. J Clin Microbiol 1996;34(8):2039–2041.

361.  Espinel-Ingroff A, Pfaller M, Messer SA, et al. Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the NCCLS M27-A2 reference method for testing new antifungal agents against clinical isolates of Candida spp. J Clin Microbiol 2004; 42(2):718–721.

362.  Espinel-Ingroff A, Pfaller M, Messer SA, et al. Multicenter comparison of the sensititre YeastOne Colorimetric Antifungal Panel with the National Committee for Clinical Laboratory standards M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcusspp., and other yeasts and yeast-like organisms. J Clin Microbiol 1999;37(3):591–595.

363.  Castro C, Serrano MC, Flores B, et al. Comparison of the Sensititre YeastOne colorimetric antifungal panel with a modified NCCLS M38-A method to determine the activity of voriconazole against clinical isolates of Aspergillus spp. J Clin Microbiol 2004;42(9):4358–4380.

364.  Morace G, Amato G, Bistoni F, et al. Multicenter comparative evaluation of six commercial systems and the National Committee for Clinical Laboratory Standards m27-a broth microdilution method for fluconazole susceptibility testing of Candida species. J Clin Microbiol2002;40(8):2953–2958.

365.  Anaissie E, Paetznick V, Bodey GP. Fluconazole susceptibility testing of Candida albicans: microtiter method that is independent of inoculum size, temperature, and time of reading. Antimicrob Agents Chemother 1991; 35(8):1641–1646.

366.  Pfaller MA, Messer SA, Coffmann S. Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870. J Clin Microbiol 1995;33(5):1094–1097.

367.  Arendrup MC, Park S, Brown S, et al. Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. Antimicrob Agents Chemother2011;55(5):1891–1895.

368.  Colombo AL, Barchiesi F, McGough DA, et al. Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal susceptibility testing. J Clin Microbiol 1995;33(3):535–540.

369.  Pfaller MA, Messer SA, Bolmstrom A, et al. Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates. J Clin Microbiol 1996;34(7):1691–1693.

370.  Pfaller MA, Messer SA, Houston A, et al. Evaluation of the Etest method for determining voriconazole susceptibilities of 312 clinical isolates of Candida species by using three different agar media. J Clin Microbiol 2000;38(10):3715–3717.

371.  Pfaller MA, Messer SA, Mills K, et al. Evaluation of Etest method for determining posaconazole MICs for 314 clinical isolates of Candida species. J Clin Microbiol 2001;39(11):3952–3954.

372.  Espinel-Ingroff A, Pfaller M, Erwin ME, et al. Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose. J Clin Microbiol1996;34(4):848–852.

373.  Espinel-Ingroff A. Etest for antifungal susceptibility testing of yeasts. Diagnostic Microbiol Infect Dis 1994; 19(4):217–220.

374.  Provine H, Hadley S. Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds. J Clin Microbiol 2000;38(2):537–541.

375.  Pfaller MA, Diekema DJ, Boyken L, et al. Evaluation of the Etest and disk diffusion methods for determining susceptibilities of 235 bloodstream isolates of Candida glabrata to fluconazole and voriconazole. J Clin Microbiol 2003;41(5):1875–1880.

376.  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(5):1289–1290.

377.  Law D, Moore CB, Denning DW. Amphotericin B resistance testing of Candida spp.: a comparison of methods. J Antimicrob Chemother 1997;40(1):109–112.

378.  Lozano-Chiu M, Paetznick VL, Ghannoum MA, et al. Detection of resistance to amphotericin B among Cryptococcus neoformans clinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies. J Clin Microbiol 1998;36(10):2817–2822.

379.  Wanger A, Mills K, Nelson PW, et al. Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Antimicrob Agents Chemother1995;39(11):2520–2522.

380.  Chaturvedi V, Ramani R, Pfaller MA. Collaborative study of the NCCLS and flow cytometry methods for antifungal susceptibility testing of Candida albicansJ Clin Microbiol 2004;42(5):2249–2251.

381.  Pfaller MA, Jones RN. Performance accuracy of antibacterial and antifungal susceptibility test methods: report from the College of American Pathologists Microbiology Surveys Program (2001-2003). Arch Pathol Lab Med 2006;130(6):767–778.

382.  Pfaller MA, Yu WL. Antifungal susceptibility testing. New technology and clinical applications. Infect Dis Clin North Am 2001;15(4):1227–1261.

383.  Arendrup MC, Cuenca-Estrella M, Lass-Florl C, et al. EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect2012;18(7):E246–E247.

384.  Lass-Florl C, Arendrup MC, Rodriguez-Tudela JL, et al. EUCAST technical note on Amphotericin B. Clin Microbiol Infect 2011;17(12):E27–E29.

385.  Arendrup MC, Cuenca-Estrella M, Donnelly JP, et al. EUCAST technical note on posaconazole. Clin Microbiol Infect 2011;17(11):E16–E17.

386.  Arendrup MC, Rodriguez-Tudela JL, Lass-Florl C, et al. EUCAST technical note on anidulafungin. Clin Microbiol Infect 2011;17(11):E18–E20.

387.  Subcommittee on Antifungal Susceptibility Testing of the ESCMID European Committee for Antimicrobial Susceptibility Testing. EUCAST technical note on voriconazole. Clin Microbiol Infect 2008;14(10):985–987.

388.  European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing. EUCAST technical note on fluconazole. Clin Microbiol Infect 2008;14(2):193–195.

389.  Subcommittee on Antifungal Susceptibility Testing of the ESCMID European Committee for Antimicrobial Susceptibility Testing. EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Clin Microbiol Infect2008;14(4):398–405.

390.  Cuenca-Estrella M, Arendrup MC, Chryssanthou E, et al. Multicentre determination of quality control strains and quality control ranges for antifungal susceptibility testing of yeasts and filamentous fungi using the methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Clin Microbiol Infect 2007;13(10):1018–1022.

391.  Cuenca-Estrella M, Lee-Yang W, Ciblak MA, et al. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of candida species. Antimicrob Agents Chemother 2002;46(11):3644–3647.

392.  Pfaller MA. Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment. Am J Med 2012;125(Suppl 1):S3–S13.

393.  Hostetler JS, Heykants J, Clemons KV, et al. Discrepancies in bioassay and chromatography determinations explained by metabolism of itraconazole to hydroxyitraconazole: studies of interpatient variations in concentrations. Antimicrob Agents Chemother 1993; 37(10):2224–2227.

394.  Perea S, Pennick GJ, Modak A, et al. Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma. Antimicrob Agents Chemother 2000;44(5):1209–1213.

395.  Cleary JD, Chapman SW, Hardin TC, et al. Amphotericin B enzyme-linked immunoassay for clinical use: comparison with bioassay and HPLC. Ann Pharmacother 1997;31(1):39–44.

396.  Decosterd LA, Rochat B, Pesse B, et al. Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin. Antimicrob Agents Chemother 2010;54(12):5303–5315.

397.  Mikami Y, Sakamoto T, Yazawa K, et al. Comparison of in vitro antifungal activity of itraconazole and hydroxy-itraconazole by colorimetric MTT assay. Mycoses 1994;37(1–2):27–33.

398.  Odds FC, Bossche HV. Antifungal activity of itraconazole compared with hydroxy-itraconazole in vitro. J Antimicrob Chemother 2000;45(3):371–373.

399.  Cendejas-Bueno E, Forastiero A, Rodriguez-Tudela JL, et al. HPLC/UV or bioassay: two valid methods for posaconazole quantification in human serum samples. Clin Microbiol Infect 2012;18(12):1229–1235.

400.  Enache-Angoulvant A, Hennequin C. Invasive Saccharomyces infection: a comprehensive review. Clin Infect Dis 2005;41(11):1559–1568.

401.  da Matta VL, de Souza Carvalho Melhem M, Colombo AL, et al. Antifungal drug susceptibility profile of Pichia anomala isolates from patients presenting with nosocomial fungemia. Antimicrob Agents Chemother 2007;51(4):1573–1576.

402.  Paphitou NI, Ostrosky-Zeichner L, Paetznick VL, et al. In vitro antifungal susceptibilities of Trichosporon species. Antimicrob Agents Chemother 2002;46(4): 1144–1146.

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