scholarly journals Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation

2020 ◽  
Vol 75 (11) ◽  
pp. 3181-3188
Author(s):  
Maria Siopi ◽  
Olga Rivero-Menendez ◽  
Georgios Gkotsis ◽  
Anthi Panara ◽  
Nikolaos S Thomaidis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensu Stricto ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Medical Settings ◽  
Nationwide Surveillance ◽  
The Mean ◽  
The One ◽  
Organically Grown ◽  
Of Greece

Abstract Background Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Methods Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. Results No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06–1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil’s load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). Conclusions This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

2019 ◽  
Vol 58 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Beatriz Bustamante ◽  
Luis Ricardo Illescas ◽  
Andrés Posadas ◽  
Pablo E Campos
Keyword(s):  
Aspergillus Fumigatus ◽  
Latin American ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Molecular Testing ◽  
Naive Patient ◽  
In Vitro Susceptibility

Abstract Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5 μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

scholarly journals Multiplecyp51A-Based Mechanisms Identified in Azole-Resistant Isolates of Aspergillus fumigatus from China

2015 ◽  
Vol 59 (7) ◽  
pp. 4321-4325 ◽  
Author(s):  
Musang Liu ◽  
Rong Zeng ◽  
Lili Zhang ◽  
Dongmei Li ◽  
Guixia Lv ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Content Type ◽  
Resistant Strains

ABSTRACTSeventy-twoA. fumigatusclinical isolates from China were investigated for azole resistance based on mutations ofcyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

scholarly journals Aspergillosis, Avian Species and the One Health Perspective: The Possible Importance of Birds in Azole Resistance

2020 ◽  
Vol 8 (12) ◽  
pp. 2037
Author(s):  
Aryse Martins Melo ◽  
David A. Stevens ◽  
Lisa A. Tell ◽  
Cristina Veríssimo ◽  
Raquel Sabino ◽  
...  
Keyword(s):  
One Health ◽  
Plant Pathogens ◽  
Bird Species ◽  
Sensu Stricto ◽  
Urban Environments ◽  
Avian Species ◽  
Azole Resistance ◽  
Natural Environments ◽  
Long Distance ◽  
The One

The One Health context considers health based on three pillars: humans, animals, and environment. This approach is a strong ally in the surveillance of infectious diseases and in the development of prevention strategies. Aspergillus spp. are fungi that fit substantially in this context, in view of their ubiquity, as well as their importance as plant pathogens, and potentially fatal pathogens for, particularly, humans and avian species. In addition, the emergence of azole resistance, mainly in Aspergillus fumigatus sensu stricto, and the proven role of fungicides widely used on crops, reinforces the need for a multidisciplinary approach to this problem. Avian species are involved in short and long distance travel between different types of landscapes, such as agricultural fields, natural environments and urban environments. Thus, birds can play an important role in the dispersion of Aspergillus, and of special concern, azole-resistant strains. In addition, some bird species are particularly susceptible to aspergillosis. Therefore, avian aspergillosis could be considered as an environmental health indicator. In this review, aspergillosis in humans and birds will be discussed, with focus on the presence of Aspergillus in the environment. We will relate these issues with the emergence of azole resistance on Aspergillus. These topics will be therefore considered and reviewed from the “One Health” perspective.

scholarly journals The one health problem of azole resistance in Aspergillus fumigatus: current insights and future research agenda

2020 ◽  
Vol 34 (4) ◽  
pp. 202-214 ◽  
Author(s):  
Paul E. Verweij ◽  
John A. Lucas ◽  
Maiken C. Arendrup ◽  
Paul Bowyer ◽  
Arjen J.F. Brinkmann ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Health Problem ◽  
Research Agenda ◽  
One Health ◽  
Future Research ◽  
Azole Resistance ◽  
Future Research Agenda ◽  
The One

scholarly journals Azole resistant Aspergillus fumigatus clinical isolate screening in azole-containing agar plates (EUCAST E.Def 10.1): low impact of plastic trays used and poor performance in cryptic species

2021 ◽  
Author(s):  
Julia Serrano-Lobo ◽  
Ana Gómez ◽  
Belén Rodríguez-Sánchez ◽  
Patricia Muñoz ◽  
Pilar Escribano ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Cryptic Species ◽  
Screening Method ◽  
Fungal Growth ◽  
Poor Performance ◽  
Sensu Stricto ◽  
Azole Resistance ◽  
Major Error ◽  
Agar Method ◽  
The Impact

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedurés performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n=91) and cryptic species (n=52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multi-dish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harbouring TR 34 -L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/L. Isolates harbouring G54R and TR 46 -Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole-resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto ; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

scholarly journals Azole Resistance in Aspergillus fumigatus Clinical Isolates from an Italian Culture Collection

2015 ◽  
Vol 60 (1) ◽  
pp. 682-685 ◽  
Author(s):  
Cristina Lazzarini ◽  
Maria Carmela Esposto ◽  
Anna Prigitano ◽  
Massimo Cogliati ◽  
Gabriella De Lorenzis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Culture Collection ◽  
Resistance Rate ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Content Type ◽  
Italian Culture

ABSTRACTThe aims of the study were to investigate the prevalence of azole resistance amongAspergillus fumigatusclinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates.

scholarly journals Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus

2018 ◽  
Author(s):  
Takashi Umeyama ◽  
Yuta Hayashi ◽  
Hisaki Shimosaka ◽  
Tatsuya Inukai ◽  
Satoshi Yamagoe ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Genome Editing ◽  
Susceptibility Testing ◽  
Genetic Recombination ◽  
Antifungal Susceptibility ◽  
Resistant Isolate ◽  
Nucleotide Sequencing ◽  
Azole Resistance ◽  
Nucleotide Polymorphisms

AbstractAzole resistance in Aspergillus fumigatus is predominantly associated with increased expression of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked to low susceptibility in azole-resistant isolates have previously been reported, few studies have been conducted to conclusively demonstrate the contribution of SNPs to decreased azole susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations Gly138Ser (GGC→AGC) and Asn248Lys (AAT→AAA) compared with the cyp51A of azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole susceptibility was slightly decreased when a Ser138 mutation was introduced into the azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole resistance.

scholarly journals Emerging aspergillosis by azole-resistant Aspergillus fumigatus at an intensive care unit in the Netherlands, 2010 to 2013

2016 ◽  
Vol 21 (30) ◽  
Author(s):  
Judith van Paassen ◽  
Anne Russcher ◽  
Astrid WM in 't Veld - van Wingerden ◽  
Paul E Verweij ◽  
Eduard J Kuijper
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
The Netherlands ◽  
Aspergillus Fumigatus ◽  
Resistance Mechanisms ◽  
Antifungal Treatment ◽  
University Hospital ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Icu Patients

The prevalence of invasive aspergillosis (IA) at the intensive care unit (ICU) is unknown and difficult to assess since IA also develops in patients lacking specific host factors. In the Netherlands, increasing azole-resistance in Aspergillus fumigatus complicates treatment of patients with IA. The aim of this study was to determine the prevalence of IA by azole-resistant A. fumigatus at the ICU among patients receiving antifungal treatment and to follow their clinical outcome and prognosis. A retrospective cohort study was conducted in a university hospital ICU from January 2010 to December 2013. From all patients who received antifungal treatment for suspected IA, relevant clinical and microbiological data were collected using a standardised questionnaire. Of 9,121 admitted ICU-patients, 136 had received antifungal treatment for suspected IA, of which 38 had a positive A. fumigatus culture. Ten of the 38 patients harboured at least one azole-resistant isolate. Resistance mechanisms consisted of alterations in the cyp51A gene, more specific TR34/L98H and TR46/T289A/Y121F. Microsatellite typing did not show clonal relatedness, though isolates from two patients were genetically related. The overall 90-day mortality of patients with IA by azole-resistant A. fumigatus and patients with suspicion of IA by azole-susceptible isolates in the ICU was 100% (10/10) vs 82% (23/28) respectively. We conclude that the changing pattern of IA in ICU patients requires appropriate criteria for recognition, diagnosis and rapid resistance tests. The increase in azole resistance rates also challenges a reconsideration of empirical antifungal therapy.

scholarly journals Azole Resistance of Environmental and Clinical Aspergillus fumigatus Isolates from Switzerland

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Arnaud Riat ◽  
Jérôme Plojoux ◽  
Katia Gindro ◽  
Jacques Schrenzel ◽  
Dominique Sanglard
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Clinical Settings ◽  
Content Type ◽  
Azole Antifungals ◽  
First Time

ABSTRACT Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target ( cyp51A ). Recently, cyp51A mutations typical for environmental azole resistance acquisition (for example, TR 34 /L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant A. fumigatus isolates of environmental origin in Switzerland with cyp51A mutations, and we show that these isolates can also be recovered from a few patients. While the TR 34 /L98H mutation was dominant, a single azole-resistant isolate exhibited a cyp51A mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment.

scholarly journals Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

2020 ◽  
Vol 6 (1) ◽  
pp. 11 ◽  
Author(s):  
Raquel Sabino ◽  
Helena Simões ◽  
Cristina Veríssimo
Keyword(s):  
Aspergillus Fumigatus ◽  
Multiplex Pcr ◽  
Real Time ◽  
Fungal Infections ◽  
Sensu Stricto ◽  
Azole Resistance ◽  
Tissue Samples ◽  
Pcr Multiplex ◽  
Resistance Patterns ◽  
Panfungal Pcr

Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.

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