A Point Mutation in the 14α-Sterol Demethylase Gene
            <i>cyp51A</i>
            Contributes to Itraconazole Resistance in
            <i>Aspergillus fumigatus</i>

Diaz-Guerra, Teresa M.; Mellado, Emilia; Cuenca‐Estrella, Manuel; Rodríguez-Tudela, Juan Luis

doi:10.1128/aac.47.3.1120-1124.2003

Cited by 254 publications

(109 citation statements)

References 26 publications

(41 reference statements)

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“…(4,(19)(20)(21)(22)(23)(24)(25)(26). For 18 isolates, no cyp51A-mediated azole resistance mechanism was found, indicating a yet-unknown mechanism causing resistance.…”

Section: Resultsmentioning

confidence: 99%

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Buil

Lee

Rijs

et al. 2017

Antimicrob Agents Chemother

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Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR 34 /L98H (11 isolates), TR 46 /Y121F/T289A (6 isolates), TR 53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.KEYWORDS VIPcheck, broth microdilution, antifungal resistance, susceptibility, azole resistance, antifungal susceptibility testing A zole resistance is an emerging problem in Aspergillus fumigatus (1), with increasing evidence that patients with azole-resistant aspergillosis fail azole therapy (2-5). Two routes of resistance selection have been signified, through patient therapy and through the exposure of A. fumigatus to azole fungicides in the environment. There are important differences between these routes of resistance selection, including patient risk factors and fungal resistance mechanisms. Resistance mechanisms that are associated with the environmental route include TR 34 /L98H and TR 46 /Y121F/T289A (6-8).Surveillance studies in The Netherlands show that of the clinical isolates that are azole resistant, between 83% and 95% harbor mutations associated with the environmental route, while approximately 15% exhibit an azole-resistant phenotype without known resistance mutations (9). As patients inhale these airborne azole-resistant spores, a resistant infection may occur in any Aspergillus disease and in patients who have never been treated with medical triazoles (3, 7, 10,...

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“…(4,(19)(20)(21)(22)(23)(24)(25)(26). For 18 isolates, no cyp51A-mediated azole resistance mechanism was found, indicating a yet-unknown mechanism causing resistance.…”

Section: Resultsmentioning

confidence: 99%

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Buil

Lee

Rijs

et al. 2017

Antimicrob Agents Chemother

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“…Although the number of patients affected is still limited (38), treatment options are clearly reduced and lead to poor outcomes (5, 39), as mutations or alterations in the cyp51A gene lead to different azole susceptibility profiles (8,16,17,20). Thus, early diagnosis and a proper antifungal prescription have direct impacts on patient survival (40).…”

Section: Discussionmentioning

confidence: 99%

“…Other emerging mechanisms of resistance have also been described, such as two point mutations (Y121F and T289A) in combination with a 46-bp TR in the promoter (13), the point mutation Y121F alone (14), or a 53-bp TR with no associated point mutations (15,16). These mutations confer different susceptibility profiles; G54 has been associated with resistance to itraconazole and posaconazole (8,17), M220 with resistance to itraconazole, high MICs for voriconazole, and variable MICs for posaconazole (10), and G448 with voriconazole resistance and some reduction in itraconazole and posaconazole susceptibility (18). TR 34 /L98H is described as being responsible for pan-azole resistance (11), TR 46 /Y121F/T289A confers resistance to posaconazole and voriconazole, with variable susceptibilities to itraconazole (19,20), and TR 53 confers resistance to voriconazole and itraconazole and lower susceptibility to posaconazole (16).…”

mentioning

confidence: 99%

“…Alteration of this gene is the major mechanism leading to azole resistance (7). Several point mutations in cyp51A have been described, although only some of them have been confirmed as being responsible for phenotypic resistance; those include mutations at positions glycine 54, methionine 220, and glycine 448 (8)(9)(10). However, the most frequent mechanism of resistance in A. fumigatus determined to date is a combination of a point mutation at position leucine 98 with a tandem repeat (TR) insertion of 34 bp in the promoter region of cyp51A (11).…”

mentioning

confidence: 99%

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Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

Bernal‐Martínez

Gil

Rivero-Menéndez

et al. 2017

Antimicrob Agents Chemother

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The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR 34 , TR 46 , and TR 53 . Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

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“…In the last few years, resistance of A. fumigatus to itraconazole has been found to be due to different mechanisms including reduced intracellular accumulation, overexpression of the target enzyme and the presence of point mutations in the cyp51A gene which codes for the target enzyme 14-α-sterol demethylase. 120,121 The number of reports of resistance of clinical isolates of A. fumigatus to voriconazole and multi-azole resistance is gradually increasing. [122][123][124] The development of resistance in an Aspergillus fumigatus isolate, originally sensitive to itraconazole and voriconazole, recovered from a case of pulmonary aspergilloma treated with voriconazole was described by sequencing of the gene with described the G448S mutation in cyp51A gene after prolonged treatment with voriconazole.…”

Section: Resistancementioning

confidence: 99%

Pharmacotherapy of Invasive Fungal Infections with Voriconazole

Ghebremedhin

2011

Clinical Medicine Reviews in Therapeutics

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Voriconazole, a second-generation and broad-spectrum triazole derivative of fluconazole, inhibits the cytochrome P450 (CYP)-dependent enzyme 14-α-sterol demethylase, which is a pivotal step in cell membrane ergosterol synthesis in fungi. CYP2C19, CYP3A4, flavin-containing monooxygenase (FMO), and to a lesser extent CYP2C9 contribute to the oxidative metabolism of voriconazole by human liver microsomes. Clinical trials have demonstrated the safety and efficacy of voriconazole for prophylaxis and treatment in candidiasis, invasive aspergillosis, and in invasive fungal infections (IFIs) caused by a variety of non-Aspergillus molds, such as Fusarium or Scedosporium spp., and was generally well tolerated as primary therapy in adults and children. The availability of both parenteral and oral formulations and the nearly complete absorption (bioavailability of 96% for adults, ca. 40% in children) of the drug after oral administration provide for ease of use and potential cost savings, and ensure that the therapeutic plasma concentrations are maintained when switching from intravenous to oral regimes. It exhibits nonlinear pharmacokinetics in healthy controls and patients at high risk of IFIs, but considerable intra-and interpatient pharmacokinetic variability has raised the question of therapeutic drug monitoring. Most common adverse events are visual disturbances and elevated transaminase levels. The emergence of resistance towards voriconazole has been rarely reported.

show abstract

A Point Mutation in the 14α-Sterol Demethylase Gene cyp51A Contributes to Itraconazole Resistance in Aspergillus fumigatus

Cited by 254 publications

References 26 publications

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

Pharmacotherapy of Invasive Fungal Infections with Voriconazole

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