Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

Selected Clinical Features of Coccidioidomycosis in Dogs.

Canine coccidioidomycosis, a systemic fungal infection endemic to arid and semiarid regions of North, Central, and South America, is commonly diagnosed in dogs living in or traveling through lower Sonoran life zones in the states of California and Arizona. Canine and human cases have geographic overlap. Similarities between clinical coccidioidomycosis in dogs and humans include asymptomatic infection, primary respiratory disease and disseminated disease. Differences include a high rate of dissemination in dogs, differences in predilection of dissemination sites, and a granulomatous or diffuse meningoencephalopathic form in the canine central nervous system (CNS) without the obstructive component seen in humans. Dogs presenting with CNS coccidioidomycosis most commonly experience seizures. Prior disease history and serology are unreliable indicators of CNS coccidioidomycosis. Magnetic resonance imaging (MRI) is advantageous for diagnosis of CNS coccidioidomycosis in dogs. Long-term administration of antifungal medication is promoted for treatment of both primary and disseminated coccidioidomycosis in dogs. Supportive treatment addressing pain, fever, inappetance, coughing, and other clinical signs improves patient care. Glucocorticoids and or anticonvulsants are also recommended for canine disseminated CNS disease. Protracted treatment times, lack of owner compliance, failure of the disease to respond to the first antifungal drug selected, and high cost are challenges of successfully treating dogs.

Current Concepts and Future Directions in the Pharmacology and Treatment of Coccidioidomycosis.

Coccidioidomycosis remains a significant clinical problem with substantial morbidity and mortality. The vast majority of infections are asymptomatic and the need for early primary therapy remains controversial. The use of triazole antifungals has improved tolerability of therapy but concerns about acute and long-term toxicities among available agents limit their use. In addition, recent findings of decreased in vitro fluconazole susceptibility to as many as 37% of Coccidioides spp. isolates raises concerns regarding optimal therapy for these infections as fluconazole is commonly used for therapy including central nervous system disease. Thus, new agents from novel antifungal classes are currently in preclinical and clinical development aimed at reducing toxicity and improving outcomes of these serious infections.

APX001 and Other Gwt1 Inhibitor Prodrugs Are Effective in Experimental Coccidioides immitis Pneumonia.

Coccidioidomycosis is a systemic fungal infection caused by the inhalation of the arthroconidia of either of two closely related dimorphic fungi, Coccidioides immitis and C. posadasii, that are endemic in the southwestern United States and other areas in the Western Hemisphere. Chronic cavitary pulmonary infections and extrapulmonary sites of infection are very difficult to treat and often require lifelong azole therapy. APX001A is the first in a new class of broad-spectrum antifungal agents that inhibit Gwt1, an enzyme which is required for cell wall localization of glycosylphosphatidylinositol (GPI)-anchored mannoproteins in fungi. APX001A and several analogs were highly active against clinical isolates of Coccidioides, inhibiting hyphal growth at low nanogram/ml concentrations. APX001 is the N-phosphonooxymethyl prodrug of APX001A, currently in clinical trials for the treatment of invasive fungal infections. Mice were treated orally once daily with 26 mg/kg/day of APX001 and the prodrug analog APX2097, 2 h after administration of the pan-cytochrome P450 inhibitor 1-aminobenzotriazole, which was used to enhance drug half-life and exposures to more closely mimic human pharmacokinetics of APX001A. Five days of treatment reduced lung colony counts by nearly 3 logs and prevented dissemination, similar to the efficacy of fluconazole dosed orally at 25 mg/kg twice daily. In a survival experiment, both APX001- and APX2097-treated mice survived significantly longer than control and fluconazole-treated mice. APX001 and other members of this new class of antifungal agents may offer great promise as effective therapies for coccidioidomycosis.

Coccidioidomycosis Complement Fixation Titer Trends in the Age of Antifungals.

Coccidioidomycosis is associated with a broad spectrum of illness severity, ranging from asymptomatic or self-limited pulmonary infection to life-threatening manifestations of disseminated disease. Serologic studies before the widespread availability of antifungals established current understanding of serologic kinetics and dynamics. Chart histories and complement fixation (CF) titer trends were analyzed for 434 antifungal-treated coccidioidomycosis patients, who were classified by three infectious disease physicians as having either pulmonary uncomplicated coccidioidomycosis (PUC) (n = 248), pulmonary chronic coccidioidomycosis (PCC) (n = 64), disseminated coccidioidomycosis (DC) not including meningitis (n = 86), or coccidioidal meningitis (CM) (n = 36). The median maximal CF titers were 1:4 for PUC patients, 1:24 for PCC patients, 1:128 for DC patients, and 1:32 for CM patients. Approximately 25.4% of PUC patients, 6.2% of PCC patients, 2.3% of DC patients, and 8.3% of CM patients did not develop detectable titers during the study period. Maximal titers developed a mean of 31 days (95% confidence interval [CI], 13 to 50 days) after initial serologic positivity, with no significant differences between groups. Serologic recurrence occurred in 9% of PUC patients, 36% of PCC patients, 50% of DC patients, and 52% of CM patients. Median titer improvement rates were 91 days/dilution for PUC patients, 112 days/dilution for PCC patients, 136 days/dilution for DC patients, and 146 days/dilution for CM patients. Receiver operating characteristic (ROC) analysis revealed that CF testing retains moderate classification value for disseminated infections (area under the curve [AUC], 0.82 [95% CI, 0.78 to 0.87]) and complicated infections (AUC, 0.82 [95% CI, 0.77 to 0.86]). A suitable cutoff value for complicated infections is ≥1:32. Findings update serologic parameters that are relevant for clinical assessment of coccidioidomycosis patients in the triazole era.

The Orotomide Olorofim Is Efficacious in an Experimental Model of Central Nervous System Coccidioidomycosis.

Olorofim (formerly F901318) is an advanced analog of the orotomide class that inhibits fungal pyrimidine biosynthesis. We evaluated the in vitro and in vivo activities of olorofim against Coccidioides species. In vitro activity was assessed against 59 clinical Coccidioides isolates. Central nervous system infections were established in mice via intracranial inoculation with Coccidioides immitis arthroconidia. Oral therapy began 48 h postinoculation and consisted of vehicle control, olorofim daily doses of 20 mg/kg (6.67 mg/kg three times daily or 10 mg/kg twice daily) or 40 mg/kg (13.3 mg/kg three times daily or 20 mg/kg twice daily), or fluconazole (25 mg/kg twice daily). Treatment continued for 7 and 14 days in the fungal burden and survival arms, respectively. Fungal burdens were assessed by CFU counts in brains. Olorofim demonstrated potent in vitro activity (MIC range, ≤0.008 to 0.06 µg/ml). Survival was significantly enhanced in mice treated with olorofim. Reductions in brain tissue fungal burdens were also observed on day 9 in the olorofim-treated groups. Improvements in survival and reductions in fungal burdens also occurred with fluconazole. More frequent dosing of olorofim was associated with enhanced survival and greater reductions in fungal burdens. In the group treated with 13.3 mg/kg olorofim three times daily, fungal burdens remained low on day 30 (15 days after treatment was stopped), with undetectable levels in 7 of 10 mice. In contrast, fungal burdens rebounded in all other groups after therapy stopped. Olorofim was highly active in vitro and in vivo against Coccidioides These results demonstrate that olorofim may have a role in the treatment of coccidioidomycosis.

Coccidioidal Meningitis: A Review on Diagnosis, Treatment, and Management of Complications.

PURPOSE OF REVIEW: This article summarizes the diagnosis and treatment of coccidioidal meningitis (CM) and its complications. An overview of current and prospective pharmacologic treatment options and monitoring parameters is provided. A consensus has not been reached regarding universally accepted therapeutic serum levels for azoles because of insufficient evidence. We describe the preferred therapeutic drug level ranges that our institution uses to monitor azole therapy. RECENT FINDINGS: Ho et al. described the preparation and administration of intrathecally delivered amphotericin B deoxycholate. Thompson et al. described possible benefits of controversial adjuvant corticosteroid therapy for secondary prevention of vasculitic infarction secondary to CM. CM was universally fatal until the advent of intrathecal amphotericin B deoxycholate therapy, the introduction of which changed the natural history of the disease in much the same way as penicillin changed the natural history of bacterial meningitis. Although there was still significant morbidity, survival rates drastically increased to approximately 70%. The introduction of azole therapy has decreased the side effects and burden of treatment but without a significant change in CM-related mortality and morbidity compared with the use of intrathecal amphotericin B deoxycholate therapy.

The Novel Fungal Cyp51 Inhibitor VT-1598 Is Efficacious in Experimental Models of Central Nervous System Coccidioidomycosis Caused by Coccidioides posadasii and Coccidioides immitis.

Coccidioidal meningitis can cause significant morbidity, and lifelong antifungal therapy is often required. VT-1598 is a fungus-specific Cyp51 inhibitor that has potent in vitro activity against Coccidioides species. We evaluated the in vivo efficacy of VT-1598 in murine models of central nervous system coccidioidomycosis caused by C. posadasii and C. immitis Infection was introduced via intracranial inoculation, and therapy began 48 h postinoculation. Oral treatments consisted of vehicle control, VT-1598, and positive controls of fluconazole in the C. immitis study and VT-1161 in the C. posadasii study. Treatment continued for 7 and 14 days in the fungal-burden and survival studies, respectively. Fungal burden was assessed in brain tissue collected 24 to 48 h posttreatment in the fungal-burden studies, on the days the mice succumbed to infection, or at prespecified endpoints in the survival studies. VT-1598 plasma concentrations were also measured in the C. posadasii study. VT-1598 resulted in significant improvements in survival in mice infected with either species. In addition, the fungal burden was significantly reduced in the fungal-burden studies. Plasma concentrations 48 h after dosing stopped remained above the VT-1598 MIC against the C. posadasii isolate, although levels were undetectable in the survival study after a 4-week washout. Whereas fungal burden remained suppressed after a 2-week washout in the C. immitis model, a higher fungal burden was observed in the survival arm of the C. posadasii model. This in vivo efficacy supports human studies to establish the utility of VT-1598 for the treatment of coccidioidomycosis.

[A case of coccidioidomycosis in Turkey imported from the United States of America]. / Türkiye'de Amerika Birlesik Devletleri kaynakli bir koksidiyoidomikoz olgusu.

Coccidioidomycosis caused by Coccidioides immitis or Coccidioides posadasii is a rare infectious disease except in endemic regions. In this report the third documented imported case of coccidioidomycosis in Turkey was presented. A thirty-year-old male patient was admitted to our hospital with fever and purulent drainage from his chest tube. He had worked in Arizona, USA, until 4 months before this presentation. While in Arizona, he experienced cough and hemoptysis and was diagnosed as pulmonary coccidioidomycosis. He was treated with itraconazole for two months and he had no symptoms for 3 years. He then returned to Turkey and 2 months after his return to Turkey, he was admitted to another hospital in Istanbul with dyspnea and diagnosed as hydro-pneumothorax, and pleural fluid obtained from the inserted chest tube was found to be purulent. One gram of BID amoxicillin-clavulanate was given. Physical examination on admission revealed a purulent drainage on the right side chest tube, a temperature of 38.5°C and decreased breath sounds on the right lung. Piperacillin-tazobactam 3 x 4.5 g intravenous and fluconazole 400 mg intravenous once daily were started. Human immunodeficiency virus test was negative. Gram-negative diplococci and rods, gram-positive cocci and septate hyphae were seen in the Gram stain of his pleural fluid. Pleural fluid culture revealed Moraxella catarrhalis after 24 hours incubation and a mold after 72 hours of incubation. Anti-coccidioidal antibodies were found positive in a titer of 1/2. Hydro-pneumothorax, atelectasis and a 3 mm nodules in the right lung were seen in his thorax CT. The patient's pleural fluid and the culture plates were sent to the Public Health Institute of Turkey, Mycology Reference Laboratory (PHIT-MRL), with a clinical suspicion of coccidioidomycosis. The specimen and plates were submitted to the PHIT-MRL Bio Safety Level-3 laboratory for mycological evaluation. The microscopic examination of 15% KOH preparations of pleural fluid specimens revealed septate hyphae which appear to be in the early stages of forming arthroconidia. The pleural fluid culture grew buff-white coloured colonies with aerial hyphae, which were suspected of being a Coccidioides spp. The strain was identified as C.immitis/posadasii by direct microscopy and culture, and subsequently confirmed by the FDA-approved DNA probe. DNA sequence analysis of the ITS and D1/D2 rDNA regions confirmed the isolate to be C.posadasii species [ITS 100% match to GenBank Accession No. AB232901 (630/630 base pair match), and D1/D2 100% match to GenBank Accession No. AB232884 (617/617 base pair match)]. ITS1 and ITS2 barcode analysis also confirmed the species to be C.posadasii, which is the species endemic in Arizona. Susceptibility testing was performed according to Clinical and Laboratory Standards Institute M38-A2 guidelines in the Fungus Testing Laboratory of the University of Texas Health Science Center at San Antonio and minimal inhibitory concentration values were; 0.125 µg/ml for amphotericin B, posaconazole and voriconazole, 0.5 µg/ml for itraconazole and 8 µg/ml for fluconazole. He had decortication of the pleura and was discharged from hospital after six weeks treatment with intravenous fluconazole which was continued orally for one year. Anti-coccidioidal antibodies were negative after two months of treatment. The patient is currently asymptomatic. The presented case is the third case reported from Turkey and provides additional contribution to the existing literature with regard to the appearance of arthroconidium, which is the unusual hyphal form, instead of the expected spherules in the infected tissue.

Divergence in the approach to tumor necrosis factor α-inhibitor recipients with coccidioidomycosis.

BACKGROUND: Tumor necrosis factor α-inhibitors (TNFIs) have been associated with increased risk of certain fungal infections, including coccidioidomycosis. The optimal treatment approach to coccidioidomycosis in TNFI recipients is unknown. METHODS: We constructed an anonymous, voluntary survey for practicing pulmonary and infectious disease physicians in the state of Arizona regarding approach to TNFI patients with coccidioidomycosis. RESULTS: There is no current consensus on managing these patients. CONCLUSIONS: Further research is necessary to determine the optimal approach to TNFI recipients with coccidioidomycosis.

Rational Design of T Lymphocyte Epitope-Based Vaccines Against Coccidioides Infection.

Coccidioidomycosis is a potentially life-threatening mycosis endemic to the Southwestern USA and some arid regions of Central and South America. A vaccine against Coccidioides infection would benefit over 30-million people who reside in or visit the endemic regions. Vaccine candidates against systemic fungal infections come in many forms. Live attenuated vaccines are derived from disease-causing pathogens and generally stimulate excellent protective immunity. Since attenuated vaccines contain living microbes, there is a degree of unpredictability raising concerns regarding safety and stability. Generation of a subunit vaccine has initiated efforts to design a safe reagent suitable for administration to humans at risk of coccidioidomycosis. Epitope-based vaccines allow for eliciting specific protective immune responses and removal of potentially detrimental sequences to improve safety. This chapter describes methods for the identification of T cell epitopes derived from Coccidioides antigens, design, and production of a recombinant vaccine containing multiple T cell epitopes, and evaluation of its protective efficacy and vaccine immunity against pulmonary Coccidioides infection using a strain of transgenic mice that express a human MHC II molecule.

Efficacy of the Investigational Antifungal VT-1161 in Treating Naturally Occurring Coccidioidomycosis in Dogs.

Coccidioidomycosis can be a chronic, systemic fungal infection requiring long-term to lifetime medication. Thus, there is a need for improved antifungal agents with greater efficacy and reduced toxicity. VT-1161 has a low affinity for mammalian cytochromes and potently inhibits fungal CYP51 with proven efficacy in murine models of central nervous system (CNS) and respiratory coccidioidomycosis. Dogs experience coccidioidomycosis similar to humans and are a useful preclinical model for naturally occurring disease. Twenty-four client-owned dogs diagnosed with respiratory coccidioidomycosis based on radiography, serology, clinical signs, and clinicopathologic abnormalities were treated with a loading dose of VT-1161 for 14 days, followed by 46 days of a lower maintenance dose. Twelve dogs received a high dose (29 mg/kg loading, 6 mg/kg maintenance) and 12 received a low dose (10 mg/kg loading, 1.6 mg/kg maintenance). Response to treatment was assessed by calculating the reduction in disease scores at exit compared to disease scores at enrollment. Overall, 20 of 24 (83%) dogs had ≥50% reduction in enrollment disease scores at exit (P < 0.001), with no difference between the high- and low-dose groups (P = 0.66). Time-weighted average plasma concentrations for the high- and low-dose groups were 39 ± 5 µg/ml and 19 ± 2 µg/ml, respectively. In this open-label study, VT-1161 was efficacious for the treatment of respiratory coccidioidomycosis in naturally infected dogs. Combined with previously reported murine data, this finding supports the further development of VT-1161 for the treatment of coccidioidomycosis in humans.

Large-Scale Evaluation of In Vitro Amphotericin B, Triazole, and Echinocandin Activity against Coccidioides Species from U.S. Institutions.

Large-scale testing of Coccidioides isolates has not been performed, and the frequency of clinical isolates with elevated amphotericin B or triazole MICs has not been evaluated. Coccidioides isolates (n = 581) underwent antifungal susceptibility testing. Elevated MIC values were observed for fluconazole (≥16 µg/ml, 37.3% of isolates; ≥32 µg/ml, 7.9% of isolates), itraconazole (≥2 µg/ml, 1.0% of isolates), posaconazole (≥1 µg/ml, 1.0% of isolates), and voriconazole (≥2 µg/ml, 1.2% of isolates). However, mold-active triazoles exhibited low MICs for the majority of isolates tested. Additional correlation with patient outcomes to determine the relevance of elevated MICs in Coccidioides isolates is needed.

Antifungal therapeutics for dimorphic fungal pathogens.

Dimorphic fungi cause several endemic mycoses which range from subclinical respiratory infections to life-threatening systemic disease. Pathogenic-phase cells of Histoplasma, Blastomyces, Paracoccidioides and Coccidioides escape elimination by the innate immune response with control ultimately requiring activation of cell-mediated immunity. Clinical management of disease relies primarily on antifungal compounds; however, dimorphic fungal pathogens create a number of challenges for antifungal drug therapy. In addition to the drug toxicity issues known for current antifungals, barriers to efficient drug treatment of dimorphic fungal infections include natural resistance to the echinocandins, residence of fungal cells within immune cells, the requirement for systemic delivery of drugs, prolonged treatment times, potential for latent infections, and lack of optimized standardized methodology for in vitro testing of drug susceptibilities. This review will highlight recent advances, current therapeutic options, and new compounds on the horizon for treating infections by dimorphic fungal pathogens.

Synthesis and in vitro antifungal activity of isoniazid-derived hydrazones against Coccidioides posadasii.

Coccidioidomycosis is a potentially severe infection caused by dimorphic fungi Coccidioides immitis and Coccidioides posadasii. Although guidelines are well established, refractory disease is a matter of concern in the clinical management of coccidioidomycosis. In the present study three isoniazid-derived hydrazones N'-[(E)-1-(4-methoxyphenyl)ethylidene]pyridine-4-carbohydrazide, N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide, and N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide were synthesized and evaluated for antifungal activity against C. posadasii. Susceptibility assays were performed by macrodilution testing. Interactions between the hydrazones and amphotericin B or itraconazole were evaluated by the checkerboard method. We also investigated the impairment of such compounds on cell ergosterol and membrane integrity. The synthesized molecules were able to inhibit C. posadasii in vitro with MIC values that ranged from 25 to 400 µg/mL. Drug interactions between synthesized molecules and amphotericin B proved synergistic for the majority of tested isolates; regarding itraconazole, synergism was observed only when strains were tested against N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide. Reduction of cellular ergosterol was observed when strains were challenged with the hydrazones alone or combined with antifungals. Only N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide altered membrane permeability of C. posadasii cells. Isoniazid-derived hydrazones were able to inhibit C. posadasii cells causing reduction of ergosterol content and alterations in the permeability of cell membrane. This study confirms the antifungal potential of hydrazones against pathogenic fungi.

Peritoneal Coccidioidomycosis: a Rare Case Report and Review of the Literature.

Coccidioidomycosis is a fungal infection endemic to the southwestern United States that typically causes a self-limited pulmonary illness. Extrapulmonary dissemination is extremely rare and typically localized to the skin, bone, and meninges. The gastrointestinal system has generally been thought to be spared from this disease. This report describes a patient who was initially diagnosed with pulmonary coccidioidomycosis with mediastinal lymphadenopathy and skin dissemination. Ten months after completion of treatment, he presented with nonspecific abdominal pain and diffuse musculoskeletal and constitutional symptoms. Radiographic imaging revealed near resolution of previously noted thoracic findings but new peritoneal thickening and enhancement suggestive of peritoneal carcinomatosis. Laparoscopic biopsies confirmed Coccidioides immitis by culture and histology without evidence of other abnormalities. This case is unique for several reasons. It is one of a relatively small number of cases that describes a diagnosis of peritoneal coccidioidomycosis and the first case identified in which a healthy patient developed extensive peritoneal disease in spite of near-complete resolution of pulmonary and skin manifestations after appropriate treatment. This case underscores the complexity of this disease and motivates more investigation into pathophysiology and treatment considerations of coccidioidomycosis in the gastrointestinal system. We will review the risk factors associated with dissemination, the interpretation of serologies, the characteristics of patients with peritoneal involvement, and finally, the current treatment guidelines.

Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models.

Coccidioidomycosis, or valley fever, is a growing health concern endemic to the southwestern United States. Safer, more effective, and more easily administered drugs are needed especially for severe, chronic, or unresponsive infections. The novel fungal CYP51 inhibitor VT-1161 demonstrated in vitro antifungal activity, with MIC50 and MIC90 values of 1 and 2 µg/ml, respectively, against 52 Coccidioides clinical isolates. In the initial animal study, oral doses of 10 and 50 mg/kg VT-1161 significantly reduced fungal burdens and increased survival time in a lethal respiratory model in comparison with treatment with a placebo (P < 0.001). Oral doses of 25 and 50 mg/kg VT-1161 were similarly efficacious in the murine central nervous system (CNS) model compared to placebo treatment (P < 0.001). All comparisons with the positive-control drug, fluconazole at 50 mg/kg per day, demonstrated either statistical equivalence or superiority of VT-1161. VT-1161 treatment also prevented dissemination of infection from the original inoculation site to a greater extent than fluconazole. Many of these in vivo results can be explained by the long half-life of VT-1161 leading to sustained high plasma levels. Thus, the efficacy and pharmacokinetics of VT-1161 are attractive characteristics for long-term treatment of this serious fungal infection.

In vitro interactions between amphotericin B and hydrocortisone: potential implications for intrathecal therapy.

Fungal meningitis remains a severe and often lethal infection requiring aggressive antifungal therapy and in refractory cases the use of intrathecal amphotericin B (AmB). Administration of amphotericin B by this method may result in clinically apparent adverse reactions such as paresthesias, radiculitis, or myelopathy. Coadministration of hydrocortisone is therefore often given in an attempt to avoid these effects; however, the potential consequences of this approach on fungal growth or on drug synergy/antagonism had not previously been assessed. We used the checkerboard titration broth microdilution method to analyze interactions by fractional inhibitory concentration indices (FICIs). The combination of amphotericin B and hydrocortisone resulted in synergy or indifference against all isolates (Candida, Cryptococcus, and Coccidioides) during in vitro testing at low concentrations. Antagonism was observed using higher hydrocortisone concentrations (those not observed in vivo) suggesting possible steric hindrance or binding to AmB may occur at doses unlikely to be present during clinical care. Concurrent hydrocortisone and AmB administration should not be avoided due to in vitro antagonism concerns.