scholarly journals Five-Year Survey (2014 to 2018) of Azole Resistance in Environmental Aspergillus fumigatus Isolates from China

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Duantao Cao ◽  
Ruilin Wu ◽  
Suxia Dong ◽  
Feiyan Wang ◽  
Chao Ju ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Soil Samples ◽  
Azole Resistance ◽  
Clinical Settings ◽  
Wild Type ◽  
Content Type

ABSTRACT A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.

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 In VitroBiochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

2015 ◽  
Vol 59 (12) ◽  
pp. 7771-7778 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Josie E. Parker ◽  
Claire L. Price ◽  
W. David Nes ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Inhibitory Concentration ◽  
Point Mutations ◽  
Residual Activity ◽  
Azole Resistance ◽  
Wild Type ◽  
Biochemical Basis ◽  
Content Type ◽  
Resistant Phenotype

ABSTRACTThe incidence of triazole-resistantAspergillusinfections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinantAspergillus fumigatusCPR1 (AfCPR1), andEscherichia colimembrane suspensions containing recombinantA. fumigatusCYP51 proteins, allowingin vitroscreening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed thatA. fumigatusCYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole thanA. fumigatusCYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance inA. fumigatusstrains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance ofA. fumigatusstrains harboring G54W, L98H, and M220K Af51A point mutations.

scholarly journals The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

2015 ◽  
Vol 59 (3) ◽  
pp. 1738-1744 ◽  
Author(s):  
Eleftheria Mavridou ◽  
Joseph Meletiadis ◽  
Antony Rijs ◽  
Johan W. Mouton ◽  
Paul E. Verweij
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Fungal Growth ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Synergistic Interactions ◽  
Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

scholarly journals Comparison of Two Typing Methods for Characterization of Azole Resistance in Aspergillus fumigatus from Potting Soil Samples in a Chinese Hospital

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Hong Fan ◽  
Yong Chen ◽  
Liping Duan ◽  
Jingya Zhao ◽  
Chunping Qin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Soil Samples ◽  
Azole Resistance ◽  
Str Typing ◽  
Content Type ◽  
Typing Methods ◽  
Close Genetic Relationship ◽  
Short Tandem ◽  
Index Of Diversity

ABSTRACT To understand the characterizations of azole resistance in Aspergillus fumigatus from potting soil samples in the hospital, a total of 58 samples were collected. Among 106 A. fumigatus samples obtained, 5 isolates from 4 soil samples located in the gerontology department were identified as azole-resistant A. fumigatus (ARAF). Four ARAF isolates harbored the TR34/L98H allele, and the other one had no mutation in the cyp51A gene. Among 174 A. fumigatus samples selected for genotyping, TRESPERG typing obtained a close discriminatory power (Simpson’s index of diversity [DI], 0.9941; 95% confidence interval [CI], 0.9913 to 0.9968) compared with the short tandem repeat (STR) typing (DI, 0.9997; 95% CI, 0.9976 to 1.0000). Genotyping showed that the TR34/L98H isolates in the hospital had a close genetic relationship with ARAF isolates from China and many other countries. In conclusion, this study indicated the presence of ARAF in potting soil samples from the hospital, which might pose a risk of causing ARAF infection in patients.

scholarly journals Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of theAspergillus fumigatus cyp51AGene in Primary Clinical Samples from Neutropenic Patients

2012 ◽  
Vol 56 (7) ◽  
pp. 3905-3910 ◽  
Author(s):  
Birgit Spiess ◽  
Wolfgang Seifarth ◽  
Natalia Merker ◽  
Susan J. Howard ◽  
Mark Reinwald ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Positive Culture ◽  
Cross Reactivity ◽  
Resistant Isolate ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Pcr Assays

ABSTRACTThe increasing incidence of azole resistance inAspergillus fumigatuscausing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediatingcyp51Agene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of theAspergillus fumigatus cyp51Agene to establish PCR assays with consecutive DNA sequence analysis to detect and identify theA. fumigatus cyp51Atandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially dilutedA. fumigatusand human DNA,A. fumigatus cyp51Agene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg ofA. fumigatusDNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons forA. fumigatuswild-type DNA confirmed thecyp51Awild-type sequence, and PCR products from one azole-resistantA. fumigatusisolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

scholarly journals The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Thaila Fernanda dos Reis ◽  
Lilian Pereira Silva ◽  
Patrícia Alves de Castro ◽  
Rafaela Andrade do Carmo ◽  
Marjorie Mendes Marini ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Mismatch Repair ◽  
Genetic Instability ◽  
Galleria Mellonella ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Azole Resistance ◽  
Repair System ◽  
Wild Type ◽  
Content Type

ABSTRACT The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatus MSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatus mshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella. Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition. IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2. Although the mutation frequency of mshA (the A. fumigatus MSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.

scholarly journals Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

2015 ◽  
Vol 53 (3) ◽  
pp. 868-874 ◽  
Author(s):  
Ga-Lai M. Chong ◽  
Wendy W. J. van de Sande ◽  
Gijs J. H. Dingemans ◽  
Giel R. Gaajetaan ◽  
Alieke G. Vonk ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Azole Resistance ◽  
Wild Type ◽  
Pcr Assay ◽  
Content Type ◽  
Resistant Strains

Azole resistance inAspergillus fumigatusis 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 relevantAspergillusspecies, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistantA. fumigatusstrains. 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 ofAspergillus. 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 ofAspergilluswas <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2Aspergillusspecies and 14A. fumigatussamples). 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 ofAspergillusspecies directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

scholarly journals Rapid Induction of Multiple Resistance Mechanisms in Aspergillus fumigatus during Azole Therapy: a Case Study and Review of the Literature

2011 ◽  
Vol 56 (1) ◽  
pp. 10-16 ◽  
Author(s):  
Simone M. T. Camps ◽  
Jan W. M. van der Linden ◽  
Yi Li ◽  
Ed J. Kuijper ◽  
Jaap T. van Dissel ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanisms ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Multiple Resistance ◽  
Review Of The Literature ◽  
Wild Type ◽  
Content Type ◽  
Type Isolate ◽  
Rapid Induction

ABSTRACTNine consecutive isogenicAspergillus fumigatusisolates cultured from a patient with aspergilloma were investigated for azole resistance. The first cultured isolate showed a wild-type phenotype, but four azole-resistant phenotypes were observed in the subsequent eight isolates. Four mutations were found in thecyp51Agene of these isolates, leading to the substitutions A9T, G54E, P216L, and F219I. Only G54 substitutions were previously proved to be associated with azole resistance. Using a Cyp51A homology model and recombination experiments in which the mutations were introduced into a susceptible isolate, we show that the substitutions at codons P216 and F219 were both associated with resistance to itraconazole and posaconazole. A9T was also present in the wild-type isolate and thus considered a Cyp51A polymorphism. Isolates harboring F219I evolved further into a pan-azole-resistant phenotype, indicating an additional acquisition of a non-Cyp51A-mediated resistance mechanism. Review of the literature showed that in patients who develop azole resistance during therapy, multiple resistance mechanisms commonly emerge. Furthermore, the median time between the last cultured wild-type isolate and the first azole-resistant isolate was 4 months (range, 3 weeks to 23 months), indicating a rapid induction of resistance.

scholarly journals In VitroAcquisition of Secondary Azole Resistance in Aspergillus fumigatus Isolates after Prolonged Exposure to Itraconazole: Presence of Heteroresistant Populations

2011 ◽  
Vol 56 (1) ◽  
pp. 174-178 ◽  
Author(s):  
Pilar Escribano ◽  
Sandra Recio ◽  
Teresa Peláez ◽  
Milagros González-Rivera ◽  
Emilio Bouza ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Tandem Repeat ◽  
Prolonged Exposure ◽  
Azole Resistance ◽  
Wild Type ◽  
Secondary Resistance ◽  
Content Type ◽  
Direct Exposure ◽  
Short Tandem

ABSTRACTSecondary resistance to azoles inAspergillus fumigatusisolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20A. fumigatusisolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in thecyp51Agene. Adjusted conidium inocula (3 × 107CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MICinitial) and after (MICfinal) exposure to itraconazole. In both procedures, the MICfinalwas significantly higher than the MICinitial. However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in thecyp51Agene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 109CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures,A. fumigatusshort tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole.

scholarly journals Contributions of both ATP-Binding Cassette Transporter and Cyp51A Proteins Are Essential for Azole Resistance in Aspergillus fumigatus

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Sanjoy Paul ◽  
Daniel Diekema ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Azole Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Gene Encoding ◽  
Pathogenic Yeast ◽  
Genetic Changes ◽  
Content Type ◽  
Transporter Protein ◽  
Atp Binding Cassette

ABSTRACT While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus. Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.

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