Molecular Identification and Susceptibility Testing of Molds Isolated in a Prospective Surveillance of Triazole Resistance in Spain (FILPOP2 Study)

ABSTRACT Antifungal resistance is increasing by the emergence of intrinsically resistant species and by the development of secondary resistance in susceptible species. A previous study performed in Spain revealed levels of azole resistance in molds of between 10 and 12.7%, but secondary resistance in Aspergillus fumigatus was not detected. We used itraconazole (ITZ)-supplemented medium to select resistant strains. A total of 500 plates supplemented with 2 mg/liter of ITZ were sent to 10 Spanish tertiary hospitals, and molecular identification and antifungal susceptibility testing were performed. In addition, the cyp51A gene in those A. fumigatus strains showing azole resistance was sequenced. A total of 493 isolates were included in the study. Sixteen strains were isolated from patients with an infection classified as proven, 104 were isolated from patients with an infection classified as probable, and 373 were isolated from patients with an infection classified as colonization. Aspergillus was the most frequent genus isolated, at 80.3%, followed by Scedosporium-Lomentospora (7.9%), Penicillium-Talaromyces (4.5%), Fusarium (2.6%), and the order Mucorales (1%). Antifungal resistance was detected in Scedosporium-Lomentospora species, Fusarium, Talaromyces, and Mucorales. Three strains of A. fumigatus sensu stricto were resistant to azoles; two of them harbored the TR34+L98H mechanism of resistance, and the other one had no mutations in cyp51A. The level of azole resistance in A. fumigatus remains low, but cryptic species represent over 10% of the isolates and have a broader but overall higher range of antifungal resistance.

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Molecular Identification, Antifungal Susceptibility Testing, and Mechanisms of Azole Resistance in Aspergillus Species Received within a Surveillance Program on Antifungal Resistance in Spain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00865-19 ◽

2019 ◽

Vol 63 (9) ◽

Cited By ~ 10

Author(s):

Olga Rivero-Menendez ◽

Juan Carlos Soto-Debran ◽

Narda Medina ◽

Jose Lucio ◽

Emilia Mellado ◽

...

Keyword(s):

Aspergillus Terreus ◽

Fungal Infections ◽

Novel Mutation ◽

Antifungal Susceptibility ◽

Resistance Mechanism ◽

Sensu Stricto ◽

Antifungal Resistance ◽

Surveillance Program ◽

Azole Resistance ◽

Content Type

ABSTRACT Antifungal resistance is one of the major causes of the increasing mortality rates for fungal infections, especially for those caused by Aspergillus spp. A surveillance program was established in 2014 in the Spanish National Center for Microbiology for tracking resistance in the most prevalent Aspergillus species. A total of 273 samples were included in the study and were initially classified as susceptible or resistant according to EUCAST breakpoints. Several Aspergillus cryptic species were found within the molecularly identified isolates. Cyp51 mutations were characterized for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus sensu stricto strains that were classified as resistant. Three A. fumigatus sensu stricto strains carried the TR34/L98H resistance mechanism, while two harbored G54R substitution and one harbored the TR46/Y121F/T289A mechanism. Seventeen strains had no mutations in cyp51A, with ten of them resistant only to isavuconazole. Three A. terreus sensu stricto strains harbored D344N substitution in cyp51A, one of them combined with M217I, and another carried an A249G novel mutation. Itraconazole-resistant A. flavus sensu stricto strains harbored P220L and H349R alterations in cyp51A and cyp51C, respectively, that need further investigation on their implication in azole resistance.

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Antifungal Resistance to Fluconazole and Echinocandins Is Not Emerging in Yeast Isolates Causing Fungemia in a Spanish Tertiary Care Center

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02670-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4565-4572 ◽

Cited By ~ 34

Author(s):

Laura Judith Marcos-Zambrano ◽

Pilar Escribano ◽

Carlos Sánchez ◽

Patricia Muñoz ◽

Emilio Bouza ◽

...

Keyword(s):

Susceptibility Testing ◽

Molecular Level ◽

Care Center ◽

Antifungal Susceptibility ◽

Tertiary Care ◽

Candida Guilliermondii ◽

Antifungal Resistance ◽

Its2 Region ◽

Content Type

ABSTRACTAccurate knowledge of fungemia epidemiology requires identification of strains to the molecular level. Various studies have shown that the rate of resistance to fluconazole ranges from 2.5% to 9% inCandidaspp. isolated from blood samples. However, trends in antifungal resistance have received little attention and have been studied only using CLSI M27-A3 methodology. We assessed the fungemia epidemiology in a large tertiary care institution in Madrid, Spain, by identifying isolates to the molecular level and performing antifungal susceptibility testing according to the updated breakpoints of European Committee for Antimicrobial Susceptibility Testing (EUCAST) definitive document (EDef) 7.2. We studied 613 isolates causing 598 episodes of fungemia in 544 patients admitted to our hospital (January 2007 to December 2013). Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region and further tested forin vitrosusceptibility to amphotericin B, fluconazole, posaconazole, voriconazole, micafungin, and anidulafungin. Resistance was defined using EUCAST species-specific breakpoints, and epidemiological cutoff values (ECOFFs) were applied as tentative breakpoints. Most episodes were caused byCandida albicans(46%),Candida parapsilosis(28.7%),Candida glabrata(9.8%), andCandida tropicalis(8%). Molecular identification enabled us to better detect cryptic species ofCandida guilliermondiiandC. parapsilosiscomplexes and episodes of polyfungal fungemia. The overall percentage of fluconazole-resistant isolates was 5%, although it was higher inC. glabrata(8.6%) and non-Candidayeast isolates (47.4%). The rate of resistance to echinocandins was 4.4% and was mainly due to the presence of intrinsically resistant non-Candidaspecies. Resistance mainly affected non-Candidayeasts. The rate of resistance to fluconazole and echinocandins did not change considerably during the study period.

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Fungemia Surveillance in Denmark Demonstrates Emergence of Non-albicans Candida Species and Higher Antifungal Usage and Resistance Rates than in Other Nations

Journal of Clinical Microbiology ◽

10.1128/jcm.01907-17 ◽

2018 ◽

Vol 56 (4) ◽

pp. e01907-17 ◽

Cited By ~ 9

Author(s):

Mariana Castanheira

Keyword(s):

Candida Albicans ◽

Candida Species ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Fungal Species ◽

Azole Resistance ◽

Content Type ◽

Fungal Organisms ◽

Echinocandin Resistance ◽

Resistance Rates

ABSTRACT Recent changes in the occurrence of fungal species and the difficulties in performing reference antifungal susceptibility testing highlight the importance of surveillance of fungal organisms and antifungal resistance rates. K. M. T. Astvad et al. report results from recent (2012 to 2015) fungemia surveillance in Denmark and compare the results to previous data (2004 to 2011), showing a decrease in Candida albicans infections accompanied by an increase in C. glabrata and C. dubliniensis infections (J Clin Microbiol 56:e01564-17, 2018, https://doi.org/10.1128/JCM.01564-17). Azole resistance among C. tropicalis and C. parapsilosis isolates and echinocandin resistance in C. krusei isolates were higher in Denmark than in other regions. Interestingly, the usage of antifungals is higher in Denmark than in other Nordic countries.

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Population-Based Survey of Filamentous Fungi and Antifungal Resistance in Spain (FILPOP Study)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00383-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3380-3387 ◽

Cited By ~ 138

Author(s):

A. Alastruey-Izquierdo ◽

E. Mellado ◽

T. Peláez ◽

J. Pemán ◽

S. Zapico ◽

...

Keyword(s):

Cryptic Species ◽

Filamentous Fungi ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Population Based ◽

Antifungal Resistance ◽

Deep Tissue ◽

Tissue Samples ◽

Content Type ◽

Population Based Survey

ABSTRACTA population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.0016/1,000 inhabitants. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus wasAspergillus, accounting for 86.3% of the isolates, followed byScedosporiumat 4.7%; the order Mucorales at 2.5%;Penicilliumat 2.2%, andFusariumat 1.2%. The most frequent species wasAspergillus fumigatus(48.5%), followed byA. flavus(8.4%),A. terreus(8.1%),A. tubingensis(6.8%), andA. niger(6.5%). Cryptic/siblingAspergillusspecies accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those ofScedosporiumand Mucorales and also among cryptic species ofAspergillus, with 40% of these isolates showing resistance to all of the antifungal compounds tested. CrypticAspergillusspecies seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.

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Species Identification andIn VitroAntifungal Susceptibility ofAspergillus terreusSpecies Complex Clinical Isolates from a French Multicenter Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02315-17 ◽

2018 ◽

Vol 62 (5) ◽

pp. e02315-17 ◽

Cited By ~ 4

Author(s):

S. Imbert ◽

A. C. Normand ◽

S. Ranque ◽

J. M. Costa ◽

J. Guitard ◽

...

Keyword(s):

Amphotericin B ◽

Antifungal Susceptibility ◽

Sensu Stricto ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Clinical Samples ◽

Azole Resistance ◽

University Hospitals ◽

Content Type ◽

Interesting Alternative

ABSTRACTAspergillussectionTerreiis a species complex currently comprised of 14 cryptic species whose prevalence in clinical samples as well as antifungal susceptibility are poorly known. The aims of this study were to investigateA. Terreiclinical isolates at the species level and to perform antifungal susceptibility analyses by reference and commercial methods. Eighty-two clinicalA. Terreiisolates were collected from 8 French university hospitals. Molecular identification was performed by sequencing parts of beta-tubulin and calmodulin genes. MICs or minimum effective concentrations (MECs) were determined for 8 antifungal drugs using both EUCAST broth microdilution (BMD) methods and concentration gradient strips (CGS). Among the 79A. Terreiisolates,A. terreus stricto sensu(n= 61),A. citrinoterreus(n= 13),A. hortai(n= 3), andA. alabamensis(n= 2) were identified. All strains had MICs of ≥1 mg/liter for amphotericin B, except for two isolates (bothA. hortai) that had MICs of 0.25 mg/liter. FourA. terreusisolates were resistant to at least one azole drug, including one with pan-azole resistance, yet no mutation in theCYP51Agene was found. All strains had low MECs for the three echinocandins. The essential agreements (EAs) between BMD and CGS were >90%, except for those of amphotericin B (79.7%) and itraconazole (73.4%). Isolates belonging to theA. sectionTerreiidentified in clinical samples show wider species diversity beyond the knownA. terreus sensu stricto. Azole resistance inside the sectionTerreiis uncommon and is not related to CYP51A mutations here. Finally, CGS is an interesting alternative for routine antifungal susceptibility testing.

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Molecular Markers of Antifungal Resistance: Potential Uses in Routine Practice and Future Perspectives

Journal of Fungi ◽

10.3390/jof7030197 ◽

2021 ◽

Vol 7 (3) ◽

pp. 197

Author(s):

Guillermo Garcia-Effron

Keyword(s):

Molecular Mechanisms ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Antifungal Resistance ◽

Routine Practice ◽

Molecular Tools ◽

Mechanisms Of Resistance ◽

Comprehensive Description ◽

Secondary Resistance ◽

Mycological Diagnosis

Antifungal susceptibility testing (AST) has come to establish itself as a mandatory routine in clinical practice. At the same time, the mycological diagnosis seems to have headed in the direction of non-culture-based methodologies. The downside of these developments is that the strains that cause these infections are not able to be studied for their sensitivity to antifungals. Therefore, at present, the mycological diagnosis is correctly based on laboratory evidence, but the antifungal treatment is undergoing a growing tendency to revert back to being empirical, as it was in the last century. One of the explored options to circumvent these problems is to couple non-cultured based diagnostics with molecular-based detection of intrinsically resistant organisms and the identification of molecular mechanisms of resistance (secondary resistance). The aim of this work is to review the available molecular tools for antifungal resistance detection, their limitations, and their advantages. A comprehensive description of commercially available and in-house methods is included. In addition, gaps in the development of these molecular technologies are discussed.

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Molecular Identification andIn VitroResponse to Antifungal Drugs of Clinical Isolates of Exserohilum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00488-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4951-4954 ◽

Cited By ~ 33

Author(s):

Keith Cássia da Cunha ◽

Deanna A. Sutton ◽

Josepa Gené ◽

Javier Capilla ◽

Josep Cano ◽

...

Keyword(s):

Sequence Analysis ◽

High Activity ◽

Molecular Identification ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Antifungal Drugs ◽

Content Type ◽

Clinical Interest ◽

Nasal Region ◽

Morphological Differences

ABSTRACTExserohilumis an agent of human and animal mycoses. Although classification has been based on a few subtle morphological differences, three species of clinical interest have been traditionally accepted. In this study, by using a multigene sequence analysis, we have demonstrated thatExserohilum longirostratumandE. mcginnisiiare probable synonyms ofE. rostratum. The isolates tested were mainly from the nasal region. Antifungal susceptibility testing demonstrated high activity of the eight agents tested against this fungus.

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Elevated MIC Values of Imidazole Drugs against Aspergillus fumigatus Isolates with TR 34 /L98H/S297T/F495I Mutation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01549-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 15

Author(s):

Yong Chen ◽

Zongwei Li ◽

Xuelin Han ◽

Shuguang Tian ◽

Jingya Zhao ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Azole Resistance ◽

Pyrenophora Teres ◽

Str Typing ◽

Content Type ◽

Resistant Strains ◽

Short Tandem

ABSTRACT The use of azole fungicides in agriculture is believed to be one of the main reasons for the emergence of azole resistance in Aspergillus fumigatus . Though widely used in agriculture, imidazole fungicides have not been linked to resistance in A. fumigatus . This study showed that elevated MIC values of imidazole drugs were observed against A. fumigatus isolates with TR 34 /L98H/S297T/F495I mutation, but not among isolates with TR 34 /L98H mutation. Short-tandem-repeat (STR) typing analysis of 580 A. fumigatus isolates from 20 countries suggested that the majority of TR 34 /L98H/S297T/F495I strains from China were genetically different from the predominant major clade comprising most of the azole-resistant strains and the strains with the same mutation from the Netherlands and Denmark. Alignments of sterol 14α-demethylase sequences suggested that F495I in A. fumigatus was orthologous to F506I in Penicillium digitatum and F489L in Pyrenophora teres , which have been reported to be associated with imidazole resistance. In vitro antifungal susceptibility testing of different recombinants with cyp51A mutations further confirmed the association of the F495I mutation with imidazole resistance. In conclusion, this study suggested that environmental use of imidazole fungicides might confer selection pressure for the emergence of azole resistance in A. fumigatus .

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Aspergillus fumigatus Clinical Isolates Carrying CYP51A with TR34/L98H/S297T/F495I Substitutions Detected after Four-Year Retrospective Azole Resistance Screening in Brazil

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02059-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 1

Author(s):

Laís Pontes ◽

Caio Augusto Gualtieri Beraquet ◽

Teppei Arai ◽

Guilherme Leite Pigolli ◽

Luzia Lyra ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Tandem Repeat ◽

Antifungal Susceptibility ◽

Antifungal Resistance ◽

Clinical Isolates ◽

Azole Resistance ◽

Resistance Screening ◽

Environmental Isolates ◽

Content Type ◽

Susceptibility Tests

ABSTRACT Azole antifungal resistance in Aspergillus fumigatus is a worldwide concern. As in most public hospitals in Brazil, antifungal susceptibility tests are not routinely performed for filamentous fungi at our institution. A 4-year retrospective azole antifungal resistance screening revealed two azole-resistant A. fumigatus clinical isolates carrying the CYP51A TR34 (34-bp tandem repeat)/L98H (change of L to H at position 98)/S297T/F495I resistance mechanism mutations, obtained from two unrelated patients. Broth microdilution antifungal susceptibility testing showed high MICs for itraconazole, posaconazole, and miconazole. Short tandem repeat (STR) typing analysis presented high levels of similarity between these two isolates and clinical isolates with the same mutations reported from the Netherlands, Denmark, and China, as well as environmental isolates from Taiwan. Our findings might indicate that active searching for resistant A. fumigatus is necessary. They also represent a concern considering that our hospital provides tertiary care assistance to immunocompromised patients who may be exposed to resistant environmental isolates. We also serve patients who receive prophylactic antifungal therapy or treatment for invasive fungal infections for years. In these two situations, isolates resistant to the antifungal in use may be selected within the patients themselves. We do not know the potential of this azole-resistant A. fumigatus strain to spread throughout our country. In this scenario, the impact on the epidemiology and use of antifungal drugs will significantly alter patient care, as in other parts of the world. In summary, this finding is an important contribution to alert hospital laboratories conducting routine microbiological testing to perform azole resistance surveillance and antifungal susceptibility tests of A. fumigatus isolates causing infection or colonization in patients at high risk for systemic aspergillosis.

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An Improved Medium for Colistin Susceptibility Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01950-17 ◽

2018 ◽

Vol 56 (5) ◽

Cited By ~ 10

Author(s):

Konrad Gwozdzinski ◽

Saina Azarderakhsh ◽

Can Imirzalioglu ◽

Linda Falgenhauer ◽

Trinad Chakraborty

Keyword(s):

Susceptibility Testing ◽

Acquired Resistance ◽

Multidrug Resistant ◽

Colistin Resistance ◽

Reliable Determination ◽

Content Type ◽

E Coli ◽

Resistant Species ◽

Mueller Hinton ◽

Serovar Typhimurium

ABSTRACTThe plasmid-located colistin resistance genemcr-1confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination ofmcr-1-containingEnterobacteriaceae. Colistin susceptibility testing was performed for 50mcr-1-containingEscherichia coliandKlebsiella pneumoniaeisolates, 7 intrinsically polymyxin-resistant species,K. pneumoniaeandE. coliisolates with acquired resistance to polymyxins due tomgrBandpmrBmutations, respectively, and 32mcr-1-negative, colistin-susceptible isolates ofAcinetobacter baumannii,Citrobacter freundii,Enterobacter cloacae,E. coli,K. pneumoniae, andSalmonella entericaserovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lackingmcr-1. Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for bothmcr-1-containingEnterobacteriaceaeisolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.

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