Dataset Information

Triazole phenotypes and genotypic characterization of clinical Aspergillus fumigatus isolates in China.

ABSTRACT: This study investigated the triazole phenotype and genotypic of clinical Aspergillus fumigatus isolates from China. We determined the triazole susceptibility profiles of 159 A. fumigatus isolates collected between 2011 and 2015 from four different areas in China tested against 10 antifungal drugs using the Clinical Laboratory Standard Institute M38-A2 method. For the seven itraconazole-resistant A. fumigatus isolates identified in the study, the cyp51A gene, including its promoter region, was sequenced and the mutation patterns were characterized. The resistant isolates were genotyped by microsatellite typing to determine the genetic relatedness to isolates from China and other countries. The frequency of itraconazole resistance in A. fumigatus isolates in our study was 4.4% (7/159). Six of the seven triazole-resistant isolates were recovered from the east and southeast of China, and one from was recovered from the west of China. No resistant isolates were found in the north. Three triazole-resistant isolates exhibited the TR34/L98H mutation, two carried the TR34/L98H/S297T/F495I mutation and one harbored a G54V mutation in the cyp51A gene. Analysis of the microsatellite markers from seven non-wild-type isolates indicated the presence of five unique genotypes, which clustered into two major genetic groups. The cyp51A gene mutations TR34/L98H and TR34/L98H/S297T were the most frequently found mutations, and the G54V mutation was reported for the first time in China. The geographic origin of the triazole-resistant isolates appeared to concentrate in eastern and south-eastern areas, which suggests that routine antifungal susceptibility testing in these areas should be performed for all clinically relevant A. fumigatus isolates to guide antifungal therapy and for epidemiological purposes.

SUBMITTER: Deng S

PROVIDER: S-EPMC5750463 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Triazole phenotypes and genotypic characterization of clinical Aspergillus fumigatus isolates in China.

Deng Shuwen S Zhang Lili L Ji Yanfeng Y Verweij Paul E PE Tsui Kin Ming KM Hagen Ferry F Houbraken Jos J Meis Jacque F JF Abliz Parida P Wang Xiaodong X Zhao Jingjun J Liao Wanqing W

Emerging microbes & infections 20171206 12

This study investigated the triazole phenotype and genotypic of clinical Aspergillus fumigatus isolates from China. We determined the triazole susceptibility profiles of 159 A. fumigatus isolates collected between 2011 and 2015 from four different areas in China tested against 10 antifungal drugs using the Clinical Laboratory Standard Institute M38-A2 method. For the seven itraconazole-resistant A. fumigatus isolates identified in the study, the cyp51A gene, including its promoter region, was se ...[more]

PMID: 29209054

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Project description:Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes.IMPORTANCEAspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1, are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals.

Project description:In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

Dataset Information

Triazole phenotypes and genotypic characterization of clinical Aspergillus fumigatus isolates in China.

Publications

Triazole phenotypes and genotypic characterization of clinical Aspergillus fumigatus isolates in China.

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