Discovery of Pyrazolone Carbothioamide Derivatives as Inhibitors of the Pdr1-KIX Interaction for Combinational Treatment of Azole-Resistant Candidiasis.

Candida glabrata has emerged as an important opportunistic pathogen of invasive candidiasis due to increasing drug resistance. Targeting Pdr1-KIX interactions with small molecules represents a potential strategy for treating drug-resistant candidiasis. However, effective Pdr1-KIX inhibitors are rather limited, hindering the validation of target druggability. Here, new Pdr1-KIX inhibitors were designed and assayed. Particularly, compound B8 possessed a new chemical scaffold and exhibited potent KIX binding affinity, leading to enhanced synergistic efficacy with fluconazole to treat resistant C. glabrata infection (FICI = 0.28). Compound B8 acted by inhibiting the efflux pump and down-regulating resistance-associated genes through blocking the Pdr1-KIX interaction. Compound B8 exhibited excellent in vitro and in vivo antifungal potency in combination with fluconazole against azole-resistant C. glabrata. It also had direct antifungal effect to treat C. glabrata infection, suggesting new mechanisms of action independent of Pdr1-KIX inhibition. Therefore, compound B8 represents a promising lead compound for antifungal drug development.

Investigation of the Defective Growth Pattern and Multidrug Resistance in a Clinical Isolate of Candida glabrata Using Whole-Genome Sequencing and Computational Biology Applications.

Candida glabrata is increasingly isolated from blood cultures, and multidrug-resistant isolates have important implications for therapy. This study describes a cholesterol-dependent clinical C. glabrata isolate (ML72254) that did not grow without blood (containing cholesterol) on routine mycological media and that showed azole and amphotericin B (AmB) resistance. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and whole-genome sequencing (WGS) were used for species identification. A modified Etest method (Mueller-Hinton agar supplemented with 5% sheep blood) was used for antifungal susceptibility testing. WGS data were processed via the Galaxy platform, and the genomic variations of ML72254 were retrieved. A computational biology workflow utilizing web-based applications (PROVEAN, AlphaFold Colab, and Missense3D) was constructed to predict possible deleterious effects of these missense variations on protein functions. The predictive ability of this workflow was tested with previously reported missense variations in ergosterol synthesis genes of C. glabrata. ML72254 was identified as C. glabrata sensu stricto with MALDI-TOF, and WGS confirmed this identification. The MICs of fluconazole, voriconazole, and amphotericin B were >256, >32, and >32 µg/mL, respectively. A novel frameshift mutation in the ERG1 gene (Pro314fs) and many missense variations were detected in the ergosterol synthesis genes. None of the missense variations in the ML72254 ergosterol synthesis genes were deleterious, and the Pro314fs mutation was identified as the causative molecular change for a cholesterol-dependent and multidrug-resistant phenotype. This study verified that web-based computational biology solutions can be powerful tools for examining the possible impacts of missense mutations in C. glabrata. IMPORTANCE In this study, a cholesterol-dependent C. glabrata clinical isolate that confers azole and AmB resistance was investigated using artificial intelligence (AI) technologies and cloud computing applications. This is the first of the known cholesterol-dependent C. glabrata isolate to be found in Turkey. Cholesterol-dependent C. glabrata isolates are rarely isolated in clinical samples; they can easily be overlooked during routine laboratory procedures. Microbiologists therefore need to be alert when discrepancies occur between microscopic examination and growth on routine media. In addition, because these isolates confer antifungal resistance, patient management requires extra care.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata.

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml-1 . In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml-1 . The results obtained from real-time PCR and analysis of the ∆∆Cq values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.

Low level of antifungal resistance of Candida glabrata blood isolates in Turkey: Fluconazole minimum inhibitory concentration and FKS mutations can predict therapeutic failure.

BACKGROUND: Candida glabrata is the third leading cause of candidaemia in Turkey; however, the data regarding antifungal resistance mechanisms and genotypic diversity in association with their clinical implication are limited. OBJECTIVES: To assess genotypic diversity, antifungal susceptibility and mechanisms of drug resistance of C glabrata blood isolates and their association with patients' outcome in a retrospective multicentre study. PATIENTS/METHODS: Isolates from 107 patients were identified by ITS sequencing and analysed by multilocus microsatellite typing, antifungal susceptibility testing, and sequencing of PDR1 and FKS1/2 hotspots (HSs). RESULTS: Candida glabrata prevalence in Ege University Hospital was twofold higher in 2014-2019 than in 2005-2014. Six of the analysed isolates had fluconazole MICs ≥ 32 µg/mL; of them, five harboured unique PDR1 mutations. Although echinocandin resistance was not detected, three isolates had mutations in HS1-Fks1 (S629T, n = 1) and HS1-Fks2 (S663P, n = 2); one of the latter was also fluconazole-resistant. All patients infected with isolates carrying HS-FKS mutations and/or demonstrating fluconazole MIC ≥ 32 µg/mL (except one without clinical data) showed therapeutic failure (TF) with echinocandin and fluconazole; seven such isolates were collected in Ege (n = 4) and Gulhane (n = 3) hospitals and six detected recently. Among 34 identified genotypes, none were associated with mortality or enriched for fluconazole-resistant isolates. CONCLUSION: Antifungal susceptibility testing should be supplemented with HS-FKS sequencing to predict TF for echinocandins, whereas fluconazole MIC ≥ 32 µg/mL may predict TF. Recent emergence of C glabrata isolates associated with antifungal TF warrants future comprehensive prospective studies in Turkey.

Acquisition of FKS2 mutation after echinocandin treatment of infective endocarditis by Candida glabrata.

Bloodstream infections caused by non-albicans Candida species are increasing and echinocandins have been extensively used especially in patients with hemodynamic instability, previous antifungal treatment and hospital risk factors for intrinsic or acquired resistance to azoles. Candida glabrata resistance to echinocandins is reported and is generally associated with previous use of echinocandins; FKS gene mutations have been associated with a worse outcome. We report the case of a 65-year-old woman who developed candidemia and endocarditis by C. glabrata with a newly acquired FKS mutation 24 months after successful treatment of infective endocarditis by C. glabrata with a double dosage of anidulafungin (200 mg daily) followed by oral voriconazole. Driven by high echinocandin MICs the strain taken by intraoperative cultures was further analyzed in a referral microbiology laboratory, confirming the new onset of point mutation S633P of the FKS2 gene.

Comparative Genomics of Serial Candida glabrata Isolates and the Rapid Acquisition of Echinocandin Resistance during Therapy.

The opportunistic pathogen Candida glabrata shows a concerning increase in drug resistance. Here, we present the analysis of two serial bloodstream isolates, obtained 12 days apart. Both isolates show pan-azole resistance and echinocandin resistance was acquired during the sampling interval. Genome sequencing identified nine nonsynonymous SNVs between the strains, including a S663P substitution in FKS2 and previously undescribed SNVs in MDE1 and FPR1, offering insight into how C. glabrata acquires drug resistance and adapts to a human host.

Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from Candida glabrata (Rec-CgHMGR) Obtained by Heterologous Expression, as a Novel Therapeutic Target Model for Testing Synthetic Drugs.

The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k m and V max for the HMG-CoA were 6.5 µM and 2.26 × 10-3 µM min-1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5 µM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.

Roles of vacuolar H+-ATPase in the oxidative stress response of Candida glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis.

Treatment of Candida glabrata cystitis remains a therapeutic challenge, and an antifungal combination using flucytosine is one option. We describe two patients with refractory C. glabrata cystitis who failed flucytosine combined with caspofungin with early-acquired high-level resistance to flucytosine due to nonsense mutations in the FUR1 gene. Rapidly acquired flucytosine resistance with microbiological failure should discourage combination of caspofungin and flucytosine during urinary candidiasis.

Candida glabrata--unique features and challenges in the clinical management of invasive infections.

Candida glabrata is a pathogenic yeast with several unique biological features. This article provides an up-to-date review on current data and reasoning aspects of this clinically problematic organism. Haploidy, absence of pseudohyphae, facultative anaerobe growth of C. glabrata, as well as its intrinsically low susceptibility to azole antifungals require specific consideration in diagnosis and treatment approaches. As C. glabrata today represents a sizeable percentage of pathogens in candidaemia, the use of azole antifungals in upfront therapy of invasive yeast infections is discouraged by recent guidelines. While the selection of C. glabrata mutants with impaired susceptibility to echinocandins has been described, analyses of several clinical studies indicate an association of improved outcomes with the use of echinocandins as the primary treatment for invasive yeast infections with potential or documented involvement of C. glabrata.

Changes in genotype and fluconazole susceptibility of isolates from patients with Candida glabrata in Tunisia.

UNLABELLED: Candida glabrata has emerged as an opportunistic pathogen of considerable importance in invasive and superficial infections. AIMS: To analyze the development of fluconazole resistance in patients under treatment through epidemiological survey in our hospital. PATIENTS AND METHODS: Twenty two patients (89 clinical strains) were collected. Molecular typing of isolates was performed by polymorphic markers. Analysis of gene expression was realized by reverse transcriptase-real time polymerase chain reactions (RT-qPCR). RESULTS: Genetic analysis showed that 63% persists with apparently unchanged strains (n=14). Among them, four showed fluconazole resistance development. A strain replacement was observed in 6 patients and two patients selected more resistant isolates during the course of treatment. An analysis of Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) and Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) expression revealed an over-expression in 10 resistant isolates. CONCLUSION: This study demonstrated that C. glabrata strain undergo frequent changes in vivo. The increase in CgCDR1 and CgCDR2 expression was the most mechanism associated with fluconazole resistance.

Abdominal candidiasis is a hidden reservoir of echinocandin resistance.

FKS mutant Candida isolates were recovered from 24% (6/25) of abdominal candidiasis patients exposed to echinocandin. Candida glabrata (29%) and Candida albicans (14%) mutants were identified. Multidrug-resistant bacteria were recovered from 83% of FKS mutant infections. Mutations were associated with prolonged echinocandin exposure (P = 0.01), breakthrough infections (P = 0.03), and therapeutic failures despite source control interventions (100%). Abdominal candidiasis is a hidden reservoir for the emergence of echinocandin-resistant Candida.

Effects of rifampin and ketoconazole on pharmacokinetics of morinidazole in healthy chinese subjects.

Morinidazole, a 5-nitroimidazole antimicrobial drug, has been approved for the treatment of amoebiasis, trichomoniasis, and anaerobic bacterial infections in China. It was reported that drug-drug interaction happened after the coadministration of ornidazole, an analog of morinidazole, and rifampin or ketoconazole. Therefore, we measured the plasma pharmacokinetics (PK) of morinidazole and its metabolites in the healthy Chinese volunteers prior to and following the administration of rifampin or ketoconazole using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The area under the concentration-time curve from time 0 to time t (AUC0-t) and maximum concentration in serum (Cmax) of morinidazole were decreased by 28% and 23%, respectively, after 6 days of exposure to 600 mg of rifampin once daily; the Cmaxs of N(+)-glucuronides were increased by 14%, while their AUC0-ts were hardly changed. After 7 days of exposure to 200 mg of ketoconazole once daily, the AUC0-t and Cmax of the parent drug were not affected significantly. Cmaxs of N(+)-glucuronides were decreased by 23%; AUC0-ts were decreased by 14%. The exposure of sulfate conjugate was hardly changed after the coadministration of rifampin or ketoconazole. Using recombinant enzyme of UGT1A9 and human hepatocytes, the mechanism of the altered PK behaviors of morinidazole and its metabolites was investigated. In human hepatocytes, ketoconazole dose dependently inhibited the formation of N(+)-glucuronides (50% inhibitory concentration [IC50], 1.5 µM), while rifampin induced the mRNA level of UGT1A9 by 28% and the activity of UGT1A9 by 53%. In conclusion, the effects of rifampin and ketoconazole on the plasma exposures of morinidazole and N(+)-glucuronide are less than 50%; therefore, rifampin and ketoconazole have little clinical significance in the pharmacokinetics of morinidazole.

Breakthrough invasive Candida glabrata in patients on micafungin: a novel FKS gene conversion correlated with sequential elevation of MIC.

Candida glabrata strains sequentially isolated from blood developed resistance to micafungin (MICs from <0.015 to 4 µg/ml). A novel mutation identified in micafungin-resistant strains at bp 262 of FKS2 (containing a deletion of F659 [F659del]) was inserted into the homologous region in FKS1.

In vitro antifungal susceptibility of Candida glabrata to caspofungin and the presence of FKS mutations correlate with treatment response in an immunocompromised murine model of invasive infection.

It has been argued that the in vitro activity of caspofungin (CSP) is not a good predictor of the outcome of echinocandin treatment in vivo. We evaluated the in vitro activity of CSP and the presence of FKS mutations in the hot spot 1 (HS1) region of the FKS1 and FKS2 genes in 17 Candida glabrata strains with a wide range of MICs. The efficacy of CSP against systemic infections from each of the 17 strains was evaluated in a murine model. No HS1 mutations were found in the eight strains showing MICs for CSP of ≤ 0.5 µg/ml, but they were present in eight of the nine strains with MICs of ≥ 1 µg/ml, i.e., three in the FKS1 gene and five in the FKS2 gene. CSP was effective for treating mice infected with strains with MICs of ≤ 0.5 µg/ml, showed variable efficacy in animals challenged with strains with MICs of 1 µg/ml, and did not work in those with strains with MICs of >1 µg/ml. In addition, mutations, including one reported for the first time, were found outside the HS1 region in the FKS2 gene of six strains with different MICs, but their presence did not influence drug efficacy. The in vitro activity of CSP was compared with that of another echinocandin, anidulafungin, suggesting that the MICs of both drugs, as well as mutations in the HS1 regions of the FKS1 and FKS2 genes, are predictive of outcome.

Recurrent arthritis by Candida glabrata, a diagnostic and therapeutic challenge.

Infectious arthritis due to Candida glabrata is very rare. A 40-year-old Iranian man had developed a painful swelling on the left knee since a year ago. A surgery (meniscectomy) was performed on his knee. However, in follow-up visit after 2 months, the patient's condition was deteriorated. Direct examination of synovial fluid with Gram and hematoxylin-eosin stains were negative for any bacterial or fungal infection or crystal elements; however, inoculation into BACTEC™ Mycosis IC/F and Plus Aerobic/F culture bottles led to the isolation of a yeast strain. The macroscopic examination on CHROMagar™ Candida medium combined with microscopical examination on CMT80 agar made a presumptive identification of the isolate to be considered as C. glabrata, and it was later on confirmed by ITS sequencing. Initial empirical treatment was started with intravenous amphotericin B for 4 weeks followed by oral itraconazole which was unsuccessful. Prescription of an oral 150-mg tablet of fluconazole was considered for a 2-month course. All symptoms completely declined, and no recurrence of infection was detected. Antifungal susceptibility testing (AFST) was performed for this isolate, and the result showed sensitivity to both amphotericin B and itraconazole and less susceptibility to fluconazole while clinical recovery was achieved by fluconazole. In any suspected clinical case caused by infectious agents, application of an effective fungal diagnostic test should be considered to avoid complications due to misdiagnosis. The correlation of AFST result with real in vivo therapeutic responses can be strain or patient dependent, and this should be considered for a successive treatment.

Caspofungin MICs correlate with treatment outcomes among patients with Candida glabrata invasive candidiasis and prior echinocandin exposure.

Mutations in Candida glabrata FKS genes, which encode the echinocandin target enzyme, are independent risk factors for treatment failures during invasive candidiasis. We retrospectively compared the ability of caspofungin susceptibility testing methods to identify C. glabrata FKS mutant isolates and predict outcomes among patients at our center. Eight percent (10/120) of sterile-site C. glabrata isolates harbored FKS1 (n = 3) or FKS2 (n = 7) mutations, including 32% (10/32) recovered from patients with prior echinocandin exposure. Median echinocandin exposures for mutant and nonmutant isolates were 55 (range, 7 to 188) and 13 (3 to 84) days, respectively (P = 0.004). Sensitivity and specificity of the CLSI caspofungin resistance breakpoint MIC (>0.12 µg/ml by broth microdilution using RPMI medium [BMD-RPMI]) were 90% (9/10) and 3% (3/110), respectively, for identifying FKS mutants. Sensitivity and specificity of receiver-operator characteristic (ROC) curve-derived breakpoints by BMD-RPMI, BMD-antibiotic medium 3, Etest, and YeastOne ranged from 70 to 100% and 89 to 95%, respectively; susceptibility rates varied from 83 to 90%. The 14-day echinocandin treatment success rate was 67% (44/66); failure was more likely with prior echinocandin exposure (P = 0.002) or infection with an FKS mutant (P = 0.0001) or echinocandin-resistant isolates by BMD-AM3, Etest, and YeastOne (P ≤ 0.03). The failure rate among patients with prior exposure and infection with a resistant isolate was 91% (10/11); it was 22% (12/55) among others (P < 0.0001). In conclusion, ROC-derived caspofungin MIC breakpoints by several methods were sensitive and specific for identifying C. glabrata FKS mutant isolates. Mutations were seen exclusively among patients with prior echinocandin exposure. A paradigm that considers prior echinocandin exposure and caspofungin MICs accurately classified treatment outcomes for C. glabrata invasive candidiasis.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment.

BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2(-ΔΔCT) method and three other software packages. The stability rankings of the reference genes by geNorm and the 2(-ΔΔCT) method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.