Antifungal susceptibility of clinical Cryptococcus gattii isolates from Colombia varies among molecular types.

Cryptococcosis by Cryptococcus gattii is endemic in Colombia, affecting mostly immunocompetent hosts. Since antifungal susceptibility differs between molecular types of cryptococcal isolates, as reported elsewhere, the aim of this study was to determine if 42 Colombian clinical isolates, VGI, VGII and VGIII, differ in the susceptibility to commonly used antifungals, using Sensititre plates. Among the molecular types, six non-wild type isolates to fluconazole, voriconazole, and 5-flucytosine, were identified. Besides, VGI and VGII were less susceptible to 5-flucytosine and azoles, respectively, than other molecular types. These findings support the applicability of practicing susceptibility testing, which could better guide treatment in cryptococcosis. LAY SUMMARY: Cryptococcosis gattii affects immunocompetent people. For a correct treatment, antifungal susceptibility testing is essential. This study shows differences in the susceptibility to commonly used antimycotics among genotypes of Colombian clinical C. gattii isolates, some of which are non-wild-type.

Anticryptococcal activity of hexane extract from Spondias tuberosa Arruda and associated cellular events.

Cryptococcosis is an opportunistic systemic mycosis whose treatment is limited to three drugs. In this work, we evaluated the antifungal activity of a hexane extract (HE) from Spondias tuberosa leaves against Cryptococcus neoformans and Cryptococcus gattii. Minimal inhibitory concentrations (MIC) were determined, and putative mechanisms were evaluated by flow cytometry. In addition, an in vivo infection assay was performed using Tenebrio molitor larvae. Treatment with HE inhibited the growth of standard and clinical isolates of C. neoformans and C. gattii (MICs ranging from 0.78 to 3.12mg/mL), significantly (P<0.05) increased mitochondrial superoxide anion levels, and induced mitochondrial membrane depolarization, loss of lysosomal membrane integrity, and phosphatidylserine externalization. The mean survival time of C. gattii-infected T. molitor larvae significantly (P<0.05) increased from 1.225 days in control to 3.067 and 3.882 days in HE-treated groups (78 and 156mg/kg, respectively). In conclusion, HE showed anticryptococcal activity, induced mitochondrial and lysosomal damage in yeast cells, and exhibited anti-infective action against C. gattii in T. molitor larvae.

Antifungal activity of promethazine and chlorpromazine against planktonic cells and biofilms of Cryptococcus neoformans/Cryptococcus gattii complex species.

Cryptococcus neoformans/Cryptococcus gattii are fungal pathogens that affect the central nervous system, mainly in immunocompromised individuals. Due to the limited pharmacological arsenal available for the treatment of cryptococcosis associated with cases of antifungal resistance of Cryptococcus spp. reported in some studies, the search for new compounds with antifungal potential becomes relevant. Thus, the objective of this study was to evaluate the inhibitory effect of phenothiazines (promethazine and chlorpromazine) on C. neoformans/C. gattii planktonic cells and biofilms. In vitro planktonic susceptibility testing was performed using the broth microdilution assay. The effect of phenothiazines was evaluated against biofilm formation and mature Cryptococcus biofilms. Biofilm morphology and ultrastructure were also evaluated by scanning electron microscopy. Promethazine and chlorpromazine showed antifungal activity against planktonic cells, with minimum inhibitory concentrations of 8-32 µg/ml and 4-16 µg/ml, respectively. As for biofilm formation, phenothiazines reduced biomass by 60% and metabolic activity by 90% at 64 µg/ml; while in mature biofilms, reductions of 85% and 90% in biomass and metabolic activity, respectively, were observed at 1024 µg/ml. Promethazine and chlorpromazine were also able to disrupt and fragment biofilms. In conclusion, promethazine and chlorpromazine have antifungal activity against planktonic cells and biofilms of Cryptococcus spp. These data show the potential of promethazine and chlorpromazine as antibiofilm drugs.

Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice from Cryptococcus gattii Independently of T Cells.

Cryptococcus neoformans causes deadly mycosis primarily in AIDS patients, whereas Cryptococcus gattii infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of C. gattii As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of C. neoformans and C. gattii infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In C. neoformans-infected mice, pICLC activity was associated with C. neoformans containment and classical Th1 immunity. In contrast, pICLC activity against C. gattii did not require any immune factors previously associated with C. neoformans immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, C. gattii pICLC activity depended on ß-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting C. gattii pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus in vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by C. gattii and C. neoformans but by distinct mechanisms; the C. gattii effect was mediated by iron limitation, while the effect on C. neoformans infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different Cryptococcus species suggests a possible mechanism by which HIV infection may select against C. gattii but not C. neoformansIMPORTANCECryptococcus neoformans and Cryptococcus gattii cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, C. gattii infects very few AIDS patients, while C. neoformans infection is an AIDS-defining illness, suggesting that the host response to HIV selects C. neoformans over C. gattii We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against C. neoformans was due to activation of well-defined immune pathways known to deter C. neoformans, whereas these immune pathways were dispensable for pICLC treatment of C. gattii Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control C. neoformans infection but is protective against C. gattii Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited C. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

Chemical Composition, Antimicrobial Activity, and Antioxidant Activity of Ocotea minarum (Nees & Mart.) Mez.

Ocotea minarum is a native plant from Brazil, popularly known as "canelinha" or "canela vassoura." The objective of this study was to investigate the chemical composition of the extracts of the bark and the leaves of O. minarum and to evaluate its antimicrobial and antioxidant activities. The phenolic compounds, flavonoids and tanins, were quantified with the reagents Folin-Ciocalteu, aluminium chloride, and vanillin. The chemical profile was performed by HPLC-DAD. The minimum inhibitory concentration was evaluated by the microdilution in a broth method. The antioxidant activity was measured by the capture of free radicals 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). In addition, protection against oxidative hemolysis and generation of malondialdehyde were evaluated in human erythrocytes. The composition of the extracts included the caffeic acid, p-coumaric acid, and rosmarinic acid, besides the flavonoids quercetin and luteolin. The EEL showed bacteriostatic action of 1000 µg/mL for all evaluated Salmonella Typhimurium, Salmonella Enteritidis, Pseudomonas aeruginosa, and Proteus mirabilis, and the EHEB had a moderate antifungal action against Candida krusei and Cryptococcus gattii (250 µg/mL). IC50 values of 8.19 (EEL) and 4.51 µg/mL (EEB) in the assay with DPPH and 6.25 (EEL) and 2.87 µg/mL (EEB) in the assay with ABTS were obtained. Up to the 3rd hour of oxidative hemolysis testing induced by AAPH, the EEB and EEL had a protective action, reducing the malondialdehyde. In conclusion, the data indicate that the O. minarum extracts can be evaluated as bioactive supplies for the development of new drugs for the prevention and treatment of diseases related to oxidative stress and microbial infections.

Pharmacological inhibition of pigmentation in Cryptococcus.

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Antifungal activity of Annona coriacea Mart. ethanol extracts against the aetiological agents of cryptococcosis.

Cryptococcosis is an opportunistic disease with a worldwide distribution. This disease is caused by fungi of the genus Cryptococcus, and its treatment is limited to several antifungals. In this study, the antifungal, cytotoxic and mutagenic properties of ethanol extracts from the bark and leaves of Annona coriacea were evaluated against the standard Cryptococcus species and clinical yeast specimens. Both extracts of A. coriacea showed inhibitory activity of 1.5 mg/mL for all of the yeasts tested. The number of viable cells at the lowest tested concentration was 0.187 mg/mL. The extracts that were tested showed inhibitory activity and reduced the fungal growth of the Cryptococcus gattii species and Cryptococcus neoformans species complexes, suggesting that this plant may be an effective alternative treatment for cryptococcosis.

Control of Cryptococcus Gattii Biofilms by an Ethanolic Extract of Cochlospermum Regium (Schrank) Pilger Leaves.

Cryptococcus gattii is an etiologic agent of cryptococcosis and a serious disease that affects immunocompromised and immunocompetent patients worldwide. The therapeutic arsenal used to treat cryptococcosis is limited to a few antifungal agents, and the ability of C. gattii to form biofilms may hinder treatment and decrease its susceptibility to antifungal agents. The objective of this study was to evaluate the antifungal and antibiofilm activities of an ethanolic extract of Cochlospermum regium (Schrank) Pilger leaves against C. gattii. The antifungal activity was assessed by measuring the minimum inhibitory concentration (MIC) using the broth microdilution technique and interaction of the extract with fluconazole was performed of checkerboard assay. The antibiofilm activity of the extract was evaluated in 96-well polystyrene microplates, and the biofilms were quantified by counting colony forming units. The extract showed antifungal activity at concentrations of 62.5 to 250 µg/mL and when the extract was evaluated in combination with fluconazole, C. gattii was inhibited at sub-MIC levels. The antibiofilm activity of the extract against C. gattii was observed both during biofilm formation and on an already established biofilm. The results showed that the ethanolic extract of the leaves of C. regium shows promise for the development of antifungal drugs to treat cryptococcosis and to combat C. gattii biofilms.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Screening of Natural Lead Molecules Against Putative Molecular Targets of Drug-resistant Cryptococcus spp: An Insight from Computer-aided Molecular Design.

Cryptococcosis is one of the major invasive fungal infections distributed worldwide with high mortality rate. C. neoformans and C. gattii are the major organisms that cause various types of infections. Anti-fungal resistances exhibited by the mentioned species of Cryptococcus threaten their effective prevention and treatment. There is limited information available on human to human transmission of the pathogen and virulent factors that are responsible for Cryptococcus mediated infections. Hence, there is high scope for understanding the mechanism, probable drug targets and scope of developing natural therapeutic agents that possess high relevance to pharmaceutical biotechnology and medicinal chemistry. The proposed review illustrates the role of computer-aided virtual screening for the screening of probable drug targets and identification of natural lead candidates as therapeutic remedies. The review initially focuses on the current perspectives on cryptococcosis, major metabolic pathways responsible for the pathogenesis, conventional therapies and associated drug resistance, challenges and scope of structure-based drug discovery. The review further illustrates various approaches for the prediction of unknown drug targets, molecular modeling works, screening of natural compounds by computational virtual screening with ideal drug likeliness and pharmacokinetic features, application of molecular docking studies and simulation. Thus, the present review probably provides AN insight into the role of medicinal chemistry and computational drug discovery to combat Cryptococcus infections and thereby open a new paradigm for the development of novel natural therapeutic against various drug targets for cryptococcal infections.

In vivo probiotic and antimicrobial photodynamic therapy as alternative therapies against cryptococcosis are ineffective.

Cryptococcosis, an invasive fungal infection distributed worldwide that affects both domestic and wild animals, has incredible rates regarding treatment failure, leading to the necessity of the development of new therapies. In this way, we aimed to evaluate the probiotic (Saccharomyces boulardii, Lactobacillus paracasei ST-11, and Lactobacillus rhamnosus GG) and antimicrobial photodynamic alternative therapies against Cryptococcus gattii in a murine model. Although previous studies suggest that these therapies can be promising against cryptococcosis, our experimental conditions for both probiotic and antimicrobial photodynamic therapies (aPDT) were not able to improve the survival of mice with cryptococcosis, even with the treatment combined with fluconazole. Our results may help other researchers to find the best protocol to test alternative therapies against Cryptococcus gattii.

Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice.

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

In vivo development of fluconazole resistance in serial Cryptococcus gattii isolates from a cat.

Elevated fluconazole minimum inhibitory concentrations (MICs) are more frequently observed in Cryptococcus gattii compared to C. neoformans isolates; however, the development of in vivo resistance and the molecular mechanisms responsible have not been reported for this species. We report a case of Cryptococcus gattii (molecular type VGIII) that developed reduced susceptibility to fluconazole during therapy and delineate the molecular mechanisms responsible. Multilocus sequence typing and quantitative DNA analysis of the pre- and post-treatment isolates was performed using well-characterized methods. Pre- and post-treatment clinical isolates were confirmed isogenic, and no differences in ERG11 or PDR11 sequences were found. qPCR found an overexpression of ERG11 and the efflux pump PDR11 in the resistant isolate compared to the isolate collected prior to initiation of antifungal therapy. Reversion to wild-type susceptibility was observed when maintained in antifungal-free media confirming the in vivo development of heteroresistance. The in vivo development of heteroresistance to fluconazole in our patient with C. gattii is secondary to overexpression of the efflux pump PDR11 and the drug target ERG11. Additional work in other clinical isolates with elevated fluconazole MICs is warranted to evaluate the frequency of heteroresistance versus point mutations as a cause of resistance.

In vitro and in vivo antifungal activities of T-2307, a novel arylamidine, against Cryptococcus gattii: an emerging fungal pathogen.

Objectives: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against the majority of fungal pathogens. In this study, the antifungal activity of T-2307 against Cryptococcus gattii was evaluated in comparison with those of amphotericin B, fluconazole and voriconazole in vitro and in vivo . Methods: The MICs for 15 clinical isolates were determined according to CLSI guidelines and time-kill studies were performed using C. gattii YF2784. In a murine model for intranasal pulmonary infection caused by C. gattii YF2784, the test compounds were administered once daily for 7 days from 2 h or 14 days post-infection. The viable counts in the lungs and brain were determined at 21 days post-infection. Results: The MIC range, MIC 50 , MIC 90 and geometric mean MIC of T-2307 were 0.0078-0.0625, 0.0313, 0.0625 and 0.0394 mg/L, respectively. The MIC of T-2307 was significantly lower than those of fluconazole, voriconazole and amphotericin B. T-2307 showed concentration-dependent fungicidal activity at 4 times the MIC or higher. Administration of T-2307 at 2 mg/kg/day, amphotericin B at 1 mg/kg/day and fluconazole at 160 mg/kg/day from 2 h post-infection significantly reduced viable counts in the lungs and brain. However, when the administration was started 14 days post-infection, only T-2307 significantly reduced the viable counts in both the lungs and the brain at 1 mg/kg/day. Conclusions: T-2307 shows excellent in vitro and in vivo antifungal activities against C. gattii and would be a promising new candidate for the treatment of cryptococcosis.

Bioactivity-guided isolation of laevicarpin, an antitrypanosomal and anticryptococcal lactam from Piper laevicarpu (Piperaceae).

Crude CH2Cl2 extract from leaves of Piper laevicarpu (Piperaceae) displayed antitrypanosomal activity against trypomastigote forms of Trypanosoma cruzi (Y strain) and antimicrobial potential against Cryptococcus gattii (strain-type WM 178). Bioactivity-guided fractionation of crude extract afforded one new natural bioactive lactam derivative, named laevicarpin. The structure of isolated compound, which displayed a very rare ring system, was elucidated based on NMR, IR and MS spectral analysis. Using MTT assay, the trypomastigotes of T. cruzi demonstrated susceptibility to laevicarpin displaying IC50 value of 14.7µg/mL (49.6µM), about 10-fold more potent than the standard drug benznidazole. The mammalian cytotoxicity of laevicarpin was verified against murine fibroblasts (NCTC cells) and demonstrated a CC50 value of 100.3µg/mL (337.7µM-SI=7). When tested against Cryptococcus gattii, laevicarpin showed an IC50 value of 2.3µg/mL (7.9µM) and a MIC value of 7.4µg/mL (25µM). Based in the obtained results, laevicarpin could be used as a scaffold for future drug design studies against the Chagas disease and anti-cryptococosis agents.

Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii.

Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.

In vitro antifungal activities of leaf extracts of Lippia alba (Verbenaceae) against clinically important yeast species.

INTRODUCTION: There are few studies reporting the antifungal activities of Lippia alba extracts. METHODS: A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. RESULTS: The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fluconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. CONCLUSIONS: The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

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.

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.