Decreased Susceptibility To Fluconazole Research Articles

Calcineurin is required for Candida glabrata Pdr1 transcriptional activation.

Fluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in C. glabrata.

Nonidentical function of Upc2A binding sites in the Candida glabrata CDR1 promoter.

Increased expression of the Candida glabrata CDR1 gene, encoding an ATP-binding cassette membrane transporter, is routinely observed in fluconazole-resistant isolates of this pathogenic yeast. CDR1 transcription has been well-documented to be due to activity of the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. Gain-of-function mutations in the gene encoding this factor are the most commonly observed cause of fluconazole hyper-resistance in clinical isolates. We have recently found that the sterol-responsive transcription factor Upc2A also acts to control CDR1 transcription, providing a direct link between ergosterol biosynthesis and expression of Pdr1 target genes. While this earlier work implicated Upc2A as an activator of CDR1 transcription, our further analyses revealed the presence of a second Upc2A binding site that negatively regulated CDR1 expression. This Upc2A binding site designated a sterol-responsive element (SRE) was found to have significant lower affinity for Upc2A DNA-binding than the previously described SRE. This new SRE was designated SRE2 while the original, positively acting site was named SRE1. A mutant version of SRE2 prevented in vitro DNA-binding by recombinant Upc2A and, when introduced into the CDR1 promoter, caused decreased fluconazole susceptibility and increased CDR1 expression. This negative effect caused by loss of SRE2 was shown to be Pdr1 independent, consistent with the presence of at least one additional activator of CDR1 transcription. The ability of Upc2A to exert either positive or negative effects on gene expression resembles behavior of mammalian nuclear receptor proteins and reveals an unexpectedly complex nature for SRE effects on gene regulation.

Clinical azole cross-resistance in Candida parapsilosis is related to a novel MRR1 gain-of-function mutation

ObjectivesHereby, we describe the molecular mechanisms underlying the acquisition of azole resistance by a Candida parapsilosis isolate following fluconazole treatment due to candiduria. MethodsA set of three consecutive C. parapsilosis isolates were recovered from the urine samples of a patient with candiduria. Whole-genome sequencing and antifungal susceptibility assays were performed. The expression of MRR1, MDR1, ERG11 and CDR1B (CPAR2_304370) was quantified by RT-qPCR. ResultsThe initial isolate CPS-A was susceptible to all three azoles tested (fluconazole, voriconazole and posaconazole); isolate CPS-B, collected after the second cycle of treatment, exhibited a susceptible-dose–dependent phenotype to fluconazole and isolate CPS-C, recovered after the third cycle, exhibited a cross-resistance profile to fluconazole and voriconazole. Whole-genome sequencing revealed a putative resistance mechanism in isolate CPS-C, associated with a G1810A nucleotide substitution, leading to a G604R change in the Mrr1p transcription factor. Introducing this mutation into the susceptible CPS-A isolate (MRR1RI) resulted in resistance to fluconazole and voriconazole, as well as up-regulation of MRR1 and MDR1. Interestingly, the susceptible-dose–dependent phenotype exhibited by isolate CPS-B was associated with an increased copy number of the CDR1B gene. The expression of CDR1B was increased in both isolates CPS-B and CPS-C and in the MRR1RI strain, harbouring the gain-of-function mutation. ConclusionsOur results describe clinical azole cross-resistance acquisition in C. parapsilosis due to a G1810A (G604R) gain-of-function mutation, resulting in MRR1 hyperactivation and consequently, MDR1 efflux pump overexpression. We also associated amplification of the CDR1B gene with decreased fluconazole susceptibility and showed that it is a putative target of the MRR1 gain-of-function mutation.

Update 2016-2018 of the Nationwide Danish Fungaemia Surveillance Study: Epidemiologic Changes in a 15-Year Perspective.

As part of a national surveillance programme initiated in 2004, fungal blood isolates from 2016–2018 underwent species identification and EUCAST susceptibility testing. The epidemiology was described and compared to data from previous years. In 2016–2018, 1454 unique isolates were included. The fungaemia rate was 8.13/100,000 inhabitants compared to 8.64, 9.03, and 8.38 in 2004–2007, 2008–2011, and 2012–2015, respectively. Half of the cases (52.8%) involved patients 60–79 years old and the incidence was highest in males ≥70 years old. Candida albicans accounted for 42.1% of all isolates and Candida glabrata for 32.1%. C. albicans was more frequent in males (p = 0.03) and C. glabrata in females (p = 0.03). During the four periods, the proportion of C. albicans decreased (p < 0.001), and C. glabrata increased (p < 0.001). Consequently, fluconazole susceptibility gradually decreased from 68.5% to 59.0% (p < 0.001). Acquired fluconazole resistance was found in 4.6% Candida isolates in 2016–2018. Acquired echinocandin resistance increased during the four periods 0.0%, 0.6%, 1.7% to 1.5% (p < 0.0001). Sixteen echinocandin-resistant isolates from 2016–2018 harboured well-known FKS resistance-mutations and one echinocandin-resistant C. albicans had an FKS mutation outside the hotspot (P1354P/S) of unknown importance. In C. glabrata specifically, echinocandin resistance was detected in 12/460 (2.6%) in 2016–2018 whereas multidrug-class resistance was rare (1/460 isolates (0.2%)). Since the increase in incidence during 2004–2011, the incidence has stabilised. In contrast, the species distribution has changed gradually over the 15 years, with increased C. glabrata at the expense of C. albicans. The consequent decreased fluconazole susceptibility and the emergence of acquired echinocandin resistance complicates the management of fungaemia and calls for antifungal drug development.

Jjj1 Is a Negative Regulator of Pdr1-Mediated Fluconazole Resistance in Candida glabrata.

The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C.glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C.glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCECandida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C.glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianusCandida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance.

Oropharyngeal Candidiasis in Palliative Care Patients in Denmark.

Oropharyngeal candidiasis (OPC) is a significant cause of morbidity, especially among patients with advanced cancer. The incidence and significance of yeast carriage and OPC in the palliative care setting in Denmark is unknown. The best diagnostic strategy and treatment regimen has to be defined. This study evaluated the clinical and microbiological incidence of yeast carriage/OPC and assessed available diagnostic procedures-culture and microscopy. The distribution of Candida species and fluconazole susceptibility was determined. Terminal care patients admitted to Hospice Sjaelland (Denmark) March to June 2012 were included. Patients were evaluated for clinical OPC and a questionnaire including previous antifungal treatment was obtained. Paired samples from the buccal mucosa and the tongue were sent to microscopy and culture. In total, 105 microscopy slides and 105 cultures from 54 patients were included, yielding 71 Candida isolates. At admission, 22% were in fluconazole treatment and 56% had received an antifungal treatment within the last month. The yeast carriage rate was 83%, whereas 48% of the patients had clinical signs of OPC. Microscopy had low positive and negative predictive value (∼50%). Candida albicans accounted for half of the isolates cultured. No C. albicans isolate displayed acquired fluconazole resistance; however, 3 out of 12 isolates of normally fluconazole-susceptible species were fluconazole resistant. These were all from patients recently treated with azoles. In total, 52% of culture-positive patients harbored at least one isolate with innately or acquired decreased fluconazole susceptibility. Therefore, susceptibility testing appears recommendable for patients with clinical signs of OPC.

Weekly use of fluconazole as prophylaxis in haematological patients at risk for invasive candidiasis.

BackgroundThe goal was to determine whether one medical centres’ unique antifungal prophylactic regimen for patients at high risk for invasive candidiasis because of their haematological malignancies, haematopoietic stem cell transplants, or high-dose chemotherapy might lead ultimately to a higher incidence of infection, to increasing fluconazole resistance, or to a shift in the predominant strain of Candida in invasive fungal episodes.MethodsData were collected retrospectively, for a ten-year period from ONKO-KISS surveillance records, and from hospital, medical, and pharmacy records and then evaluated with respect to incidence of fungal infection episodes, emergence of antifungal drug resistance, and predominance of specific Candida strains in isolate cultures. Fisher’s exact test and linear regression were used to compare minimum inhibitory concentrations and to compare the incidence of different Candida isolates, respectively.ResultsThe incidence of infection remained quite stable over 10 years with a median of 0.67 episodes/1000 bed days. Overall, Candida glabrata was the predominant species with 29% followed by C. albicans and C. krusei (14% each). No significant increment of non-albicans Candida species with decreased fluconazole susceptibility was perceived over this decade.ConclusionsOnce weekly administration of 400 mg of fluconazole to prevent candidaemia appears to have no negative impact on the efficacy as a prophylaxis when compared to standard of care (400 mg of fluconazole daily).Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0573-5) contains supplementary material, which is available to authorized users.

Species distribution and susceptibility profile to fluconazole, voriconazole and MXP-4509 of 551 clinical yeast isolates from a Romanian multi-centre study.

This is the first multi-centre study regarding yeast infections in Romania. The aim was to determine the aetiological spectrum and susceptibility pattern to fluconazole, voriconazole and the novel compound MXP-4509. The 551 isolates were identified using routine laboratory methods, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and DNA sequence analysis. Susceptibility testing was performed using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) method and breakpoints. The yeasts originated from superficial infections (SUP, 51.5 %), bloodstream infections (BSI, 31.6 %) and deep-seated infections (DEEP, 16.9 %), from patients of all ages. Nine genera and 30 species were identified. The 20 Candida species accounted for 94.6 % of all isolates. C. albicans was the overall leading pathogen (50.5 %). Lodderomyces elongisporus is reported for the first time as a fungaemia cause in Europe. C. glabrata and Saccharomyces cerevisiae, as well as the non-Candida spp. and non-albicans Candida spp. groups, showed decreased fluconazole susceptibility (<75 %). The overall fluconazole resistance was 10.2 %. C. krusei accounted for 27 of the 56 fluconazole-resistant isolates. The overall voriconazole resistance was 2.5 % and was due mainly to C. glabrata and C. tropicalis isolates. Fluconazole resistance rates for the three categories of infection were similar to the overall value; voriconazole resistance rates differed: 4 % for BSI, 3.2 % for DEEP and 1.4 % for SUP. The antifungal activity of MXP-4509 was superior to voriconazole against C. glabrata and many fluconazole-resistant isolates. There was a large percentage of non-albicans Candida isolates. A large part of the high fluconazole resistance was not acquired but intrinsic, resulting from the high percentage of C. krusei.

Gain-of-Function Mutations in UPC2 Are a Frequent Cause of ERG11 Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documented UPC2 gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressed ERG11 by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.

The Changing Epidemiology of Oropharyngeal Candidiasis in Patients with HIV/AIDS in the Era of Antiretroviral Therapy.

The impact of antiretroviral therapy (ART) on opportunistic conditions in HIV patients continues to evolve. We specifically studied the changing epidemiology of oropharyngeal candidiasis (OPC) in 215 HIV/AIDS patients. Status of yeast colonization was assessed from oral rinse samples, and preliminary yeast identification was made using CHROMagar Candida and confirmed with standard microbiological techniques and/or molecular sequencing. Susceptibility to fluconazole was determined by CHROMagar Candida agar dilution screening and CLSI broth microdilution. 176 (82%) patients were colonized and 59 (27%) patients had symptomatic OPC. Candida albicans was the most prevalent species, though C. glabrata and C. dubliniensis were detected in 29% of isolates. Decreased fluconazole susceptibility occurred in 10% of isolates. Previous ART reduced the risk of OPC, while smoking increased the risk of colonization. Oral yeast colonization and symptomatic infection remain common even with advances in HIV therapy. C. albicans is the most common species, but other yeasts are prevalent and may have decreased susceptibility to fluconazole.

Clinical and economic outcomes of decreased fluconazole susceptibility in patients with Candida glabrata bloodstream infections

The impact of reduced fluconazole susceptibility on clinical and economic outcomes in patients with Candida glabrata bloodstream infections (BSI) is unknown. A retrospective cohort study was conducted to evaluate 30-day inpatient mortality and postculture hospital charges in patients with C glabrata BSI with decreased fluconazole susceptibility (minimum inhibitory concentration [MIC] ≥ 16 μg/mL) versus fluconazole-susceptible C glabrata BSI (MIC ≤ 8 μg/mL). These analyses were adjusted for demographics, comorbidities, and time at risk. Secondary analyses limited the C glabrata group with decreased fluconazole susceptibility to MIC ≥ 64 μg/mL. There were 45 (31%) deaths among 144 enrolled patients: 19 deaths (25%) among 76 patients with C glabrata BSI with decreased fluconazole susceptibility and 26 deaths (38%) among 68 patients with fluconazole-susceptible C glabrata BSI. Decreased fluconazole susceptibility was not independently associated with increased 30-day inpatient mortality (adjusted odds ratio, .60; 95% confidence interval (CI): .26-1.35; P = 0.22) or hospital charges (multiplicative change in hospital charges, .93; 95% CI: .60-1.43; P = 0.73). Older age was associated with increased mortality and increased time at risk was associated with increased hospital charges. Crude mortality rates remain high in patients with C glabrata BSI. However, decreased fluconazole susceptibility was not associated with increased mortality or hospital charges.

High- versus low-dose fluconazole therapy for empiric treatment of suspected invasive candidiasis among high-risk patients in the intensive care unit: a cost-effectiveness analysis

ABSTRACTBackground: High-dose fluconazole is an alternative for patients with candidemia caused by Candida glabrata or other Candida species with decreased fluconazole susceptibility. However, empiric high-dose fluconazole is not currently recommended and may result in higher drug costs and toxicity.Objective: To determine the cost-effectiveness of using empiric high-dose fluconazole in intensive care unit (ICU) with suspected invasive candidiasis.Design: Decision analytic model. Target population: ICU patients with suspected invasive candidiasis. Time horizon: Lifetime. Perspective: Societal.Interventions: Low-dose fluconazole (loading dose of 800 mg followed by 400 mg daily) vs. high-dose fluconazole (loading dose of 1600 mg followed by 800 mg daily). Generic fluconazole costs were used for the analysis. Outcome measures: Incremental life expectancy and incremental cost per discounted life year (DLY) saved.Result of base-case analysis: Based on current national levels of fluconazole resistance and ability to correctly identify patients with candidemia, high-dose fluconazole was the more effective but more expensive treatment strategy. Empiric high-dose fluconazole therapy decreased the mortality rate by 0.15% compared to low-dose strategy with a cost-effectiveness rate of $55 526 per DLY saved.Results of sensitivity analysis: Empirical high-dose fluconazole was an acceptable treatment strategy (using $100 000 per DLY saved as threshold) unless the physical age of an ICU survivor was 66 years or older. Empirical high-dose fluconazole was an acceptable treatment strategy using $50 000 per DLY saved with minor changes in parameters estimates.Limitations: The estimates of our model may not be applicable to all ICU patients. Other hospitals with differences in fluconazole resistance, prevalence of invasive candidiasis, or duration of fluconazole therapy may produce different results.Conclusion: These results suggest that empiric high-dose fluconazole therapy should reduce the mortality associated with invasive candidiasis at an acceptable cost.

Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis.

Molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme and increased efflux of drug, but the impact of specific treatment regimens on resistance has not been established. A patient with advanced AIDS was enrolled in a longitudinal study to receive continuous oral fluconazole (FLU) 200 mg/day for the treatment of oropharyngeal candidosis (OPC). Oral cultures were obtained at time of enrollment, during episodes of OPC and quarterly for surveillance. The patient had five symptomatic relapses on continuous FLU during 43 months. All OPC episodes were successfully treated with increasing doses of FLU although increased FLU MICs were detected for C. albicans isolates with progression of time. DNA-typing techniques demonstrated that resistance developed in a persistent strain of C. albicans. Both FLU-resistant and isogenic isolates with reduced susceptibility were detected in the same clinical samples through multiple episodes. Analysis of molecular mechanisms of resistance revealed overexpression of MDR and CDR genes encoding efflux pumps (but not ERG11) in isolates with decreased FLU susceptibility. In addition, the presence of the G464S amino acid substitution in their lanosterol demethylase, affecting its affinity for FLU, was also detected. However, other isogenic, but FLU-susceptible isolates recovered from the same samples did not harbour the mutation, indicating microevolution of yeast populations within the oral cavity. In this patient, the continuous antifungal pressure exerted by FLU resulted in development of resistance of multifactorial nature. Despite their clonal origin, different subpopulations of C. albicans demonstrated distinct resistance mechanisms, including concomitant presence and absence of functional point mutations in ERG11 genes.

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.

Resistance to fluconazole is becoming an increasing problem in the management of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Strains obtained from five patients developed decreased fluconazole susceptibility over time. DNA strain typing confirmed the high degree of relatedness among isolates from one patient and the variability among isolates from different patients. Expression of genes involved in development of fluconazole resistance was monitored in each isolate using probes specific for ERG11 (lanosterol 14alpha-demethylase), MDR1 (a major facilitator), and CDR (ATP-binding cassette or ABC transporter) genes. Increased expression of CDR genes was detected in the series of isolates from two patients. Isolates from one of the two patients also demonstrated increased ERG11 expression, whereas isolates from the other patient did not. Increased levels of MDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression of MDR1 with subsequent overexpression of CDR genes and a final isolate again overexpressing MDR1 were detected in serial isolates from another patient. In another patient, overexpression of these genes was not detected despite an eightfold increase in fluconazole MIC. In this patient, sequence data of the ERG11 gene revealed no point mutations associated with decreased susceptibility. Five different patterns of gene expression were observed in isolates recovered from five patients who developed resistance. Therefore, these experiments demonstrate that a variety of mechanisms or combinations of mechanisms are associated with the development of fluconazole drug resistance. Additional studies are needed to estimate the frequency and clinical impact of these mechanisms of resistance.