Molecular Mechanisms of Fluconazole Resistance in Candida dubliniensis Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

ABSTRACT Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

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Distinct Patterns of Gene Expression Associated with Development of Fluconazole Resistance in Serial Candida albicansIsolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.11.2932 ◽

1998 ◽

Vol 42 (11) ◽

pp. 2932-2937 ◽

Cited By ~ 147

Author(s):

Jose L. Lopez-Ribot ◽

Robert K. McAtee ◽

Linda N. Lee ◽

William R. Kirkpatrick ◽

Theodore C. White ◽

...

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

Sequence Data ◽

Point Mutations ◽

Oropharyngeal Candidiasis ◽

Fluconazole Resistance ◽

Expression Of Genes ◽

Immunodeficiency Virus ◽

Major Facilitator ◽

Abc Transporter Genes

ABSTRACT 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 14α-demethylase), MDR1 (a major facilitator), andCDR (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 ofMDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression ofMDR1 with subsequent overexpression of CDRgenes 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 theERG11 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.

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Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in Candida albicans Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2676-2684.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2676-2684 ◽

Cited By ~ 314

Author(s):

Sofia Perea ◽

José L. López-Ribot ◽

William R. Kirkpatrick ◽

Robert K. McAtee ◽

Rebecca A. Santillán ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Candida Albicans ◽

Molecular Mechanisms ◽

Azole Resistance ◽

Mechanisms Of Resistance ◽

Fluconazole Resistance ◽

Immunodeficiency Virus ◽

Genes Encoding ◽

High Level ◽

Target Enzyme

ABSTRACT Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles inC. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.

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Detection of Candida dubliniensis in Oropharyngeal Samples from Human Immunodeficiency Virus-Infected Patients in North America by Primary CHROMagar Candida Screening and Susceptibility Testing of Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.36.10.3007-3012.1998 ◽

1998 ◽

Vol 36 (10) ◽

pp. 3007-3012 ◽

Cited By ~ 112

Author(s):

William R. Kirkpatrick ◽

Sanjay G. Revankar ◽

Robert K. Mcatee ◽

Jose L. Lopez-Ribot ◽

Annette W. Fothergill ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Germ Tube ◽

Susceptibility Testing ◽

San Antonio ◽

Antifungal Susceptibility ◽

Tube Formation ◽

Candida Dubliniensis ◽

Differential Growth ◽

Immunodeficiency Virus ◽

Dark Green

Candida dubliniensis has been associated with oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV). C. dubliniensis isolates may have been improperly characterized as atypical Candida albicans due to the phenotypic similarity between the two species. Prospective screening of oral rinses from 63 HIV-infected patients detected atypical dark green isolates on CHROMagar Candida compared to typical C. albicans isolates, which are light green. Forty-eight atypical isolates and three control strains were characterized by germ tube formation, differential growth at 37, 42, and 45°C, identification by API 20C, fluorescence, chlamydoconidium production, and fingerprinting by Ca3 probe DNA hybridization patterns. All isolates were germ tube positive. Very poor or no growth occurred at 42°C with 22 of 51 isolates. All 22 poorly growing isolates at 42°C and one isolate with growth at 42°C showed weak hybridization of the Ca3 probe with genomic DNA, consistent with C. dubliniensis identification. No C. dubliniensisisolate but only 18 of 28 C. albicans isolates grew at 45°C. Other phenotypic or morphologic tests were less reliable in differentiating C. dubliniensis from C. albicans. Antifungal susceptibility testing showed fluconazole MICs ranging from ≤0.125 to 64 μg/ml. Two isolates were resistant to fluconazole (MIC, 64 μg/ml) and one strain was dose dependent susceptible (MIC, 16 μg/ml). MICs of other azoles, including voriconazole, itraconazole, and SCH 56592, for these isolates were lower. C. dubliniensis was identified in 11 of 63 (17%) serially evaluated patients. Variability in phenotypic characteristics dictates the use of molecular and biochemical techniques to identifyC. dubliniensis. This study identifies C. dubliniensis in HIV-infected patients from San Antonio, Tex., and shows that C. dubliniensis is frequently detected in those patients by using a primary CHROMagar screen.

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Resistance Mechanisms in Clinical Isolates of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1704-1713.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1704-1713 ◽

Cited By ~ 299

Author(s):

Theodore C. White ◽

Scott Holleman ◽

Francis Dy ◽

Laurence F. Mirels ◽

David A. Stevens

Keyword(s):

Candida Albicans ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Point Mutations ◽

Resistance Mechanisms ◽

Northern Blotting ◽

Clinical Isolates ◽

Cross Resistance ◽

Polymorphism Analysis ◽

Mechanisms Of Resistance

ABSTRACT Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.

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Multiple Resistant Phenotypes of Candida albicansCoexist during Episodes of Oropharyngeal Candidiasis in Human Immunodeficiency Virus-Infected Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1621 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1621-1630 ◽

Cited By ~ 67

Author(s):

Jose L. Lopez-Ribot ◽

Robert K. McAtee ◽

Sofia Perea ◽

William R. Kirkpatrick ◽

Michael G. Rinaldi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Dna Typing ◽

Etiologic Agent ◽

Oropharyngeal Candidiasis ◽

Mechanisms Of Resistance ◽

Geometric Means ◽

Antifungal Drug Resistance ◽

Immunodeficiency Virus ◽

Serial Episodes ◽

Lanosterol Demethylase

ABSTRACT Mechanisms of resistance to azoles in Candida albicans, the main etiologic agent of oropharyngeal candidiasis (OPC), include alterations in the target enzyme (lanosterol demethylase) and increased efflux of drug. Previous studies on mechanisms of resistance have been limited by the fact that only a single isolate from each OPC episode was available for study. Multiple isolates from each OPC episode were evaluated with oral samples plated in CHROMagar Candida with and without fluconazole to maximize detection of resistant yeasts. A total of 101 isolates from each of three serial episodes of OPC from four different patients were evaluated. Decreasing geometric means of fluconazole MICs with serial episodes of infection were detected in the four patients. However, 8-fold or larger (up to 32-fold) differences in fluconazole MICs were detected within isolates recovered at the same time point in 7 of 12 episodes. Strain identity was analyzed by DNA typing techniques and indicated that isolates from each patient represented mainly isogenic strains, but differed among patients. A Northern blot technique was used to monitor expression ofERG11 (encoding lanosterol demethylase) and genes coding for efflux pumps. This analysis revealed that clinical isolates obtained from the same patient and episode were phenotypically heterogeneous in their patterns of expression of these genes involved in fluconazole resistance. These results demonstrate the complexity of the distribution of the molecular mechanisms of antifungal drug resistance and indicate that different subpopulations of yeasts may coexist at a given time in the same patient and may develop resistance through different mechanisms.

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Talaromyces atroroseus in HIV and non-HIV patient: A first report from Indonesia

Medical Mycology ◽

10.1093/mmy/myz090 ◽

2019 ◽

Vol 58 (4) ◽

pp. 560-563

Author(s):

Sem Samuel Surja ◽

Robiatul Adawiyah ◽

Jos Houbraken ◽

Anna Rozaliyani ◽

Ridhawati Sjam ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amphotericin B ◽

Molecular Analysis ◽

Room Temperature ◽

Antifungal Susceptibility ◽

Molecular Study ◽

Susceptibility Test ◽

Clinical Isolates ◽

Immunodeficiency Virus ◽

Antifungal Susceptibility Test

Abstract We performed morphology, molecular study and antifungal susceptibility test on 10 Talaromyces sp. isolates: eight clinical isolates (human immunodeficiency virus (HIV) and non-HIV-patient) and two isolates from rats. All strains produced red soluble pigment and microscopically showed Penicillium-like structure in room temperature and yeast-like structure in 37°C. Based on molecular analysis, nine isolates were identified as Talaromyces atroroseus (including the isolates from rats) and one as T. marneffei. Our susceptibility result of T. marneffei supports the use of amphotericin B, itraconazole for talaromycosis marneffei management. Talaromyces atroroseus showed variable MIC to echinocandin, azole derivatives, 5-flucytosine and amphotericin B.

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