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Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing.

ABSTRACT: A real-time PCR and pyrosequencing method was developed to detect Aspergillus fumigatus in whole blood by amplifying the cyp51A gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole.

SUBMITTER: Trama JP

PROVIDER: S-EPMC548089 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing.

Trama Jason P JP Mordechai Eli E Adelson Martin E ME

Journal of clinical microbiology 20050201 2

A real-time PCR and pyrosequencing method was developed to detect Aspergillus fumigatus in whole blood by amplifying the cyp51A gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole. ...[more]

PMID: 15695702

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In vitro susceptibility of Aspergillus fumigatus to isavuconazole: correlation with itraconazole, voriconazole, and posaconazole.

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The Effect of Posaconazole, Itraconazole and Voriconazole in the Culture Medium on Aspergillus fumigatus Triazole Resistance.

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Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole.

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RNA-Seq of Aspergillus fumigatus mutants resistant to itraconazole, SYBARIS project.

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2016-04-30 | E-SYBR-5 | biostudies-arrayexpress

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

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Project description:Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

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tpo3 and dur3, Aspergillus fumigatus Plasma Membrane Regulators of Polyamines, Regulate Polyamine Homeostasis and Susceptibility to Itraconazole.

Project description:Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections, which have high mortality rates in immunosuppressed individuals. Long-term antifungal drug azole use in clinical treatment and agriculture results in loss of efficacy or drug resistance. Drug resistance is related to cellular metabolites and the corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics under itraconazole (ITC) treatment, we identified two plasma membrane-localized polyamine regulators tpo3 and dur3, which were important for polyamine homeostasis and susceptibility to ITC in A. fumigatus. In the absence of tpo3 and/or dur3, the levels of cytoplasmic polyamines had a moderate increase, which enhanced the tolerance of A. fumigatus to ITC. In comparison, overexpression of tpo3 or dur3 induced a drastic increase in polyamines, which increased the sensitivity of A. fumigatus to ITC. Further analysis revealed that polyamines concentration-dependently affected the susceptibility of A. fumigatus to ITC by scavenging reactive oxygen species (ROS) at a moderate concentration and promoting the production of ROS at a high concentration rather than regulating drug transport. Moreover, inhibition of polyamine biosynthesis reduced the intracellular polyamine content, resulted in accumulation of ROS and enhanced the antifungal activity of ITC. Interestingly, A. fumigatus produces much lower levels of ROS under voriconazole (VOC) treatment than under ITC-treatment. Accordingly, our study established the link among the polyamine regulators tpo3 and dur3, polyamine homeostasis, ROS content, and ITC susceptibility in A. fumigatus.

| S-EPMC7772357 | biostudies-literature

Aftpo3 and Afdur3, the Aspergillus fumigatus Plasma Membrane Transporters of Polyamines, Regulate Polyamine Homeostasis and Itraconazole Susceptibility

Project description:Aspergillus fumigatus is a notorious opportunistic pathogen that causes Invasive Aspergillosis (IA) infections with high mortality in immunosuppressed individuals. Long-term antifungal drug azole abuse in clinical treatment and agriculture renders it ineffectual or drug resistant. Drug resistance can relate to the cellular metabolites and corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics following itraconazole (ITC) treatment we identified two plasma membrane-localized polyamine transporters Aftpo3 and Afdur3, which were important for polyamine homeostasis and ITC susceptibility in A. fumigatus. In the absence of Aftpo3 or Afdur3, the levels of cytoplasm polyamines had a moderate increase, accompanied by resistance to ITC. In comparison, overexpression of Aftpo3 or Afdur3 induced drastic increase of polyamines, which were linked with the sensitivity to ITC. Accordingly, our study demonstrated that concentration-dependence of polyamines affected susceptibility of A. fumigatus to ITC, by scavenging reactive oxygen species (ROS) at a moderate concentration or generating ROS at a high concentration rather than drug transport.

2020-12-21 | MTBLS1739 | MetaboLights

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