Prevalence Of Azole Resistance Research Articles

Distribution of Aspergillus species and prevalence of azole resistance in clinical and environmental samples from a Spanish hospital during a three-year study period.

Surveillance studies are crucial for updating trends in Aspergillus species and antifungal susceptibility information. Determine the Aspergillus species distribution and azole resistance prevalence during this 3-year prospective surveillance study in a Spanish hospital. Three hundred thirty-five Aspergillus spp. clinical and environmental isolates were collected during a 3-year study. All isolates were screened for azole resistance using an agar-based screening method and resistance was confirmed by EUCAST antifungal susceptibility testing. The azole resistance mechanism was confirmed by sequencing the cyp51A gene and its promoter. All Aspergillus fumigatus strains were genotyped using TRESPERG analysis. Aspergillus fumigatus was the predominant species recovered with a total of 174 strains (51.94%). The rest of Aspergillus spp. were less frequent: Aspergillus niger (14.93%), Aspergillus terreus (9.55%), Aspergillus flavus (8.36%), Aspergillus nidulans (5.37%) and Aspergillus lentulus (3.28%), among other Aspergillus species (6.57%). TRESPERG analysis showed 99 different genotypes, with 72.73% of the strains being represented as a single genotype. Some genotypes were common among clinical and environmental A. fumigatus azole-susceptible strains, even when isolated months apart. We describe the occurrence of two azole-resistant A. fumigatus strains, one clinical and another environmental, that were genotypically different and did not share genotypes with any of the azole-susceptible strains. Aspergillus fumigatus strains showed a very diverse population although several genotypes were shared among clinical and environmental strains. The isolation of azole-resistant strains from both settings suggest that an efficient analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus.

The C2H2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus.

Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination.IMPORTANCEAspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

P081 Azole-resistant Aspergillus fumigatus among NIH hospitalized patients with underlying primary immunodeficiencies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Aspergillus fumigatus causes a variety of diseases in humans. The drugs recommended for treatment of Aspergillus diseases are the mold-active azole antifungals. However, a wide range of mutations in A. fumigatus confers azole resistance, which commonly involves modifications in the cyp51Agene, the target for azole antifungal drugs.MethodsWe investigated 255 clinical A. fumigatus isolates obtained from patients hospitalized at National Institutes of Health Clinical Center, Bethesda, Maryland, USA. The species-level identification of each isolate was evaluated by colony morphology, microscopic characteristics, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and PCR-sequencing of the β-tubulin gene. We also studied sequence-based analysis of the Cyp51A gene for the azole-resistant isolates.The azole antifungal susceptibility profile of each isolate was initially evaluated using 4-well triazole screen plates (Microbiology Associates LLC, Rockville, MD, USA) containing itraconazole (4 μg/ml), voriconazole (2 μg/ml), posaconazole (0.5 μg/ml), and growth control. The full array of antifungal susceptibility was confirmed using microbroth dilution method according to Clinical and Laboratory Standards Institute CLSI M38-A3 guidelines.ResultsOf 255 A. fumigatus isolates, 12 grew on the wells containing azoles, indicating an azole-resistant phenotype. The results were read and recorded after 24 and 48 h of incubation at 35-37°C. Majority of our isolates had visible growth at 24 h. Sequence analysis of the CYP51A gene indicated the presence of M220K mutation in all 12 isolates and no mutations in the other isolates.The fact that the azole resistance was found in A. fumigatus isolated from patients with previous azole exposure, underscores the possibility that prevalence of azole-resistance might be underestimated in various patient populations because in-vitro susceptibility testing of A. fumigatus is not routinely performed.ConclusionIn conclusion, prevalence of azole resistance in clinical A. fumigatus isolates obtained from NIH patients underlying primary immunodeficiencies was 4.7%; all the resistant isolates exhibited azole-resistance mutation in Cyp51A gene.Our finding adds to the growing list of regions where acquired resistance in A. fumigatus of is documented. Our results also indicate that 4-well triazole screen plates are a reliable tool for azole-resistance screening and the selection of isolates that require a full panel of antifungal susceptibility testing.

Distribution of Aspergillus Species and Prevalence of Azole Resistance in Respiratory Samples From Swiss Tertiary Care Hospitals.

Among 400 Aspergillus species from respiratory samples in Switzerland, Aspergillus fumigatus was the most frequent species. Non-fumigatus Aspergillus spp were more prevalent among solid organ transplant recipients and after azole exposure. Azole resistance was detected in 4 A fumigatus isolates, 3 of them with the “environmental” mutation TR34/L98H in the cyp51A gene.

A Low-Cost Tebuconazole-Based Screening Test for Azole-Resistant Aspergillus fumigatus.

The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least-developed countries. Here, we present and validate a low-cost screening protocol that can be used to differentiate between azole-susceptible "wild-type" and azole-resistant A. fumigatus isolates. © 2020 The Authors. Basic Protocol 1: Preparation of Tebucheck multi-well plates Basic Protocol 2: Inoculation of Tebucheck multi-well plates.

High prevalence of triazole-resistant Aspergillus fumigatus sensu stricto in an Argentinean cohort of patients with cystic fibrosis.

Triazole resistance in Aspergillus fumigatus sensu stricto due to mutations in the cyp51A gene has been widely reported. Data from Argentina, and particularly from cystic fibrosis (CF) patients, are limited. To investigate the prevalence and molecular mechanisms of azole resistance in A. fumigatus sensu stricto recovered from this population. Ninety-three A. fumigatus isolates from 50 CF patients were retrospectively analysed for azole resistance using the standard microbroth dilution method according to CLSI M38-A2 guidelines. Sequencing analysis of the cyp51A gene and its promoter region was conducted in those isolates displaying high MIC values to itraconazole, voriconazole and/or posaconazole. Overall, 14% of isolates displayed high MIC values to at least one azole. Of them, 30.7% had the mutation TR34-L98H. No mutations in the cyp51A gene or its promoter were found in the remaining non-wild-type strains. Therefore, other mechanisms associated with azole resistance can be highly prevalent in these isolates. To the best of our knowledge, this is the first study in Latin America reporting azole-resistant A. fumigatus strains recovered from respiratory secretions of CF patients. Noteworthy, the prevalence of azole resistance in A. fumigatus sensu stricto in the studied Argentinean CF population is alarmingly high.

Fungicide-driven alterations in azole-resistant Aspergillus fumigatus are related to vegetable crops in Colombia, South America

ABSTRACTAspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR34/L98H and TR46/Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the β-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR46/Y121F/T289A (n = 8), TR34/L98H, and TR53. Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Prospective survey of azole drug resistance among environmental and clinical isolates of Aspergillus fumigatus in a French University hospital during major demolition works

ObjectivesRecent studies have reported the emerging worldwide problem of azole drug resistance of A. fumigatus isolates. The aim of this study was to evaluate the antifungal susceptibilities of A. fumigatus isolates recovered from air and clinical samples collected in a French University hospital (Lyon), which underwent major deconstruction works over a one year-period. MethodsA daily surveillance of fungal contamination was implemented during 11-months. Environmental survey was realized by air samplings, outdoor and indoor, with an automatic agar sampler. In parallel, surveillance of IA infection cases was conducted by epidemiological investigation. Environmental and clinical isolates of A. fumigatus were identified by conventional methods and β-tubulin sequencing. Susceptibility testing of A. fumigatus isolates against Itraconazole (ITZ), Voriconazole (VCZ) was performed using Etest method. ResultsA total of 3885 air samples (1744 outdoor samples and 2141 indoor samples) were collected. From the 3073 identified colonies of A. fumigatus, 400 A. fumigatus isolates were tested for their susceptibility to ITZ and VCZ, including 388 isolates coming from the environment (indoor n:157, outdoor n:231) and 12 isolates coming from clinical samples. All the 400 isolates were susceptible to azoles (≤1μg/mL). ConclusionsNo environmental reservoir of A. fumigatus azole resistant strains was found in our hospital which was undergoing major demolition works. Further studies with larger number of A. fumigatus clinical isolates and environmental isolates from agricultural areas and healthcare establishments are needed to better appreciate the occurrence and prevalence of azole resistance.

Low Prevalence of Azole Resistance in Aspergillus fumigatus in Australia, and Molecular Characterisation of-Resistant Isolates

Low Prevalence of Azole Resistance in Aspergillus fumigatus in Australia, and Molecular Characterisation of-Resistant Isolates

Azole-resistant Aspergillus fumigatus with TR34/L98H mutations in the cyp51A gene in Iran

Introduction Triazole antifungal drugs such as itraconazole, posaconazole and voriconazole are the mainstay of therapy in the management and prophylaxis of aspergillosis. However, in recent years, triazole-resistant clinical isolates of Aspergillus fumigatus have emerged in Europe (the Netherlands, Denmark, Spain, UK, Belgium, Germany and France) and Asia (China, India) resulting in several cases of therapeutic failure caused by triazole-resistant A. fumigatus. Azole resistance may develop in patients who are treated with long-term azole therapy or may develop in the environment through the exposure of the fungus to the azole fungicides used in agriculture. Methods In the present study, we investigated the presence of azole resistance in environmental and clinical A. fumigatus isolated both from hospital surroundings area and infected patients. Results In vitro antifungal susceptibility testing has shown high MICs of itraconazole (> 16 mg/L) and voriconazole (> 8 mg/L). The TR34/L98H mutation was the only resistance mechanism with no mutation in G54, M220, G138C, T289A, Y121F and G432C. Azole-resistant A. fumigatus isolates had the same TR34/L98H STRAf genotype with Netherlands and India. It is emphasized that routine resistance surveillance studies focusing on environmental and clinical samples are warranted to yield the true prevalence of azole resistance in A. fumigatus in Iran. Conclusion The widespread application of triazole fungicides in the environment in Iran could have contributed to the emergence of environmental azole-resistant A. fumigatus. Given the emergence of azole resistance in environmental strains, continued surveillance of resistance in clinical A. fumigatus strains in Iran is desirable for successful therapy of aspergillosis. Furthermore, the need for enhanced understanding of the evolution of azole resistance and measures to prevent the emergence of multiple-azole-resistant A. fumigatus strains in countries using fungicides can hardly be over-emphasized.

First determination of azole resistance in Aspergillus fumigatus strains carrying the TR34/L98H mutations in Turkey.

Aspergillus fumigatus is the most important etiological agent of invasive aspergillosis. Recently, an increasing number of azole-resistant A.fumigatus isolates have been described in various countries. The prevalence of azole resistance was investigated in this study using our culture collection of A.fumigatus isolates collected between 1999 and 2012 from clinical specimens. Seven hundred and forty-six A.fumigatus isolates, collected from 419 patients, were investigated. First, all isolates were screened for resistance to itraconazole by subculturing on Sabouraud dextrose agar that contained 4mg/L itraconazole. For isolates that grew on the itraconazole containing agar, the invitro activities of amphotericin B, itraconazole, voriconazole and posaconazole were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A reference method. After PCR amplification, the full sequence of the cyp51A gene and its promoter region was determined for all invitro azole-resistant isolates. Itraconazole resistance was found in 10.2% of the A.fumigatus isolates. From 2000 onwards, patients were observed annually with an itraconazole-resistant isolate. According to invitro susceptibility tests, amphotericin B exhibited good activity against all isolates whereas the azoles were resistant. Sequence analysis of the promoter region and CYP51A gene indicated the presence of TR34/L98H in 86.8% (n=66) of isolates. This initial analysis of the resistance mechanism of A.fumigatus from Turkey revealed a common TR34/L98H mutation in the cyp51A gene.

Azole-ResistantAspergillus fumigatus, Iran

Azole-Resistant<i>Aspergillus fumigatus</i>, Iran

The impact of azole resistance on aspergillosis guidelines

Azole resistance in Aspergillus species may be on the rise, with significant potential implications for the management of invasive aspergillosis. The main mechanism of azole resistance in Aspergillus fumigatus is via alterations of the target enzyme CYP51A. Such azole resistance is either primary or secondary (in the setting of prior azole exposure) and can be derived either from single or multiple mutations. Irrespective of the amino acid substitution type in CYP51A, azole-resistant Aspergillus isolates are always itraconazole resistant. There is significant variability among studies and centers in the prevalence of azole resistance, and this is a multifactorial issue. Nevertheless, the exact frequency of azole resistance is unknown, in part because of the low culturability of the fungus in patients with aspergillosis. This work aims to provide an overview of the current knowledge in Aspergillus azole resistance and raises questions for future research and practical implications in the management of aspergillosis.

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole-naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide-driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole-resistant Aspergillus diseases.

Azole Antifungal Resistance Today: Focus on Aspergillus

Oral triazole therapy is well established for the treatment of invasive aspergillosis (IPA), allergic aspergillosis (ABPA), and chronic pulmonary aspergillosis (CPA), and is often long-term. Resistance to triazole azole antifungal drugs in Aspergillus fumigatus is now a major clinical problem in a number of European locations, in China, Canada and the USA with particularly high frequencies from the north-west of the UK, and The Netherlands. A number of centers are reporting the continuing increasing frequency and evolution of resistance mechanisms in A. fumigatus, in both azole-naïve and patients treated with azoles. The increasing rate of resistance is of concern. A number of resistance mechanisms have been found. The biofilm modality of Aspergillus growth may have a number of therapeutic implications for aspergillosis, including antifungal resistance. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. Direct resistance testing in culture-negative clinical samples may add additional insights into the prevalence of azole resistance in A. fumigatus.

Comment on: Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies

Azole resistance is an emerging clinical problem in patients with Aspergillus infections, primarily those caused by Aspergillus fumigatus. Alanio et al. 1 describe the results of a study that investigated the prevalence of azole resistance in 118 clinical Aspergillus isolates obtained from a cohort of 89 patients with haematological malignancy. They found only one azole-resistant A. fumigatus isolate, which corresponded to a prevalence of 0.85%. 1 Although the low prevalence of resistance may be reassuring, the circumstances surrounding the resistant isolate are a cause of great concern. The azole-resistant isolate was cultured from an azole-naive patient, which precludes resistance development through azole therapy. The only other possibility is that the patient acquired the resistant isolate from the environment, as A. fumigatus is …

An observational study on the epidemiological and mycological profile of Candidemia in ICU patients

SummaryBackgroundThis study aimed to analyze the epidemiological and mycological profile of candidemia in intensive care unit (ICU) patients attending a tertiary care teaching hospital in the Himalayan region of northern India.Material/MethodsA 15-bed medico-surgical ICU and a 5-bed pediatric ICU. Ninety-one consecutively admitted ICU patients were screened for the presence of candidemia by performing blood cultures at periodic intervals.ResultsThe recovered Candida isolates were speciated and subjected to antifungal susceptibility testing using standard procedures. Forty-one of the recruited patients (45%) were found to be candidemic, with the majority of patients being in the extremes of age (13 neonates and 15 >65 years of age). Four risk factors were found to be significantly associated with the occurrence of candidemia in our patients – a period of hospitalization exceeding 7 days (p=0.0008), previous use of antibiotics (p=0.001), presence of chronic renal failure (p=0.003), and ongoing cancer chemotherapy (p= 0.041). Ninety-six Candida isolates were recovered from the 41 culture-positive patients, with Candida albicans being the commonest isolate recovered (n=75, 78.1%), followed by Candida tropicalis (n=15, 16%), and Candida glabrata (n=6, 6.5%). Fluconazole resistance was observed among 26% of all Candida isolates and 17.3% of C. albicans isolates.ConclusionsContrary to the majority of recent reports, species shift towards non-albicans candidemia has not been observed in our center, though the prevalence of azole resistance is alarmingly high even among the C. albicans isolates.

Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism

BackgroundResistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.Methods and FindingsWe investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.ConclusionsAzole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.