An expanded Agar‐Based Screening method for azole resistant Aspergillus fumigatus

Mycoses ◽  
2021 ◽  
Author(s):  
Jose Lucio ◽  
Irene Gonzalez‐Jimenez ◽  
Rocio Garcia‐Rubio ◽  
Maria Soledad Cuetara ◽  
Emilia Mellado
Keyword(s):  
Aspergillus Fumigatus ◽  
Screening Method

scholarly journals Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
L. Bernal-Martínez ◽  
H. Gil ◽  
O. Rivero-Menéndez ◽  
S. Gago ◽  
M. Cuenca-Estrella ◽  
...  
Keyword(s):  
High Resolution ◽  
Aspergillus Fumigatus ◽  
Tandem Repeats ◽  
High Resolution Melting ◽  
Screening Method ◽  
Melting Curve ◽  
Health Concern ◽  
Azole Resistance ◽  
Content Type ◽  
Selection Of

ABSTRACT The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

scholarly journals Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azole-resistant Aspergillus fumigatus

2017 ◽  
Vol 72 (12) ◽  
pp. 3325-3333 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Paul E Verweij ◽  
Johan W Mouton ◽  
Katrien Lagrou ◽  
Joseph Meletiadis
Keyword(s):  
Aspergillus Fumigatus ◽  
Screening Method

scholarly journals Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
J. B. Buil ◽  
H. A. L. van der Lee ◽  
A. J. M. M. Rijs ◽  
J. Zoll ◽  
J. A. M. F. Hovestadt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensitivity And Specificity ◽  
Antifungal Susceptibility ◽  
Screening Method ◽  
Point Mutations ◽  
Azole Resistance ◽  
Minimal Growth ◽  
Content Type ◽  
Mean Sensitivity ◽  
Selection Of

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

scholarly journals Absence of Azole Antifungal Resistance in Aspergillus fumigatus Isolated from Root Vegetables Harvested from UK Arable and Horticultural Soils

2020 ◽  
Vol 6 (4) ◽  
pp. 208
Author(s):  
Mireille H. van der Torre ◽  
Cheryl Whitby ◽  
Christopher P. Eades ◽  
Caroline B. Moore ◽  
Lilyann Novak-Frazer ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Antifungal Susceptibility ◽  
Geographical Area ◽  
Screening Method ◽  
Significant Risk ◽  
Reference Method ◽  
Resistance Mechanisms ◽  
Indoor Environments ◽  
Sample Type ◽  
Root Vegetables

The emergence of azole-resistant Aspergillus fumigatus (ARAf) complicates the treatment of aspergillosis and can nearly double the mortality from invasive aspergillosis (IA). ARAf has been isolated from many different environmental sites and indoor environments and thus presents a significant risk for susceptible patients. Local surveillance of environmental ARAf can guide antifungal prescribing and improve patient outcomes. In this study, seventy-four soils samples collected from the surface of a variety of root vegetables from farm shops and private gardens covering a wide geographical area of the UK, were cultured to assess the presence of A. fumigatus, and the prevalence and nature of any resistance mechanisms. A high-throughput in-house antifungal susceptibility screening method was developed and validated using the EUCAST MIC reference method, E.DEF 9.3.1. A total of 146 isolates were recovered and analysed. Even though the study premise was that soil-covered root vegetables and other fresh produce could represent a conduit for ARAf exposure in vulnerable patients, no ARAf were found in the soil samples despite 55% of samples harbouring A. fumigatus. The sample type and screening method used could be suitable for more extensive monitoring of the soil to detect trends in the prevalence of ARAf.

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 Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azole-resistant Aspergillus fumigatus

2018 ◽  
Vol 73 (8) ◽  
pp. 2274-2274 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Paul E Verweij ◽  
Johan W Mouton ◽  
Katrien Lagrou ◽  
Joseph Meletiadis
Keyword(s):  
Aspergillus Fumigatus ◽  
Screening Method

scholarly journals Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis

2008 ◽  
Vol 74 (22) ◽  
pp. 7023-7035 ◽  
Author(s):  
Matthias Brock ◽  
Grégory Jouvion ◽  
Sabrina Droin-Bergère ◽  
Olivier Dussurget ◽  
Marie-Anne Nicola ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Light Emission ◽  
Screening Method ◽  
Growth Conditions ◽  
Antifungal Drugs ◽  
In Vivo Studies ◽  
Drug Efficiency

ABSTRACT Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.

An integrated screening method for evaluating the pulmonary toxicity of inhaled particulates: Role of microscopic techniques in assessing pulmonary macrophage clearance functions

1990 ◽  
Vol 48 (3) ◽  
pp. 826-827
Author(s):  
David B. Warheit ◽  
Lena Achinko ◽  
Mark A. Hartsky
Keyword(s):  
Bronchoalveolar Lavage ◽  
Pulmonary Toxicity ◽  
Screening Method ◽  
Pulmonary Response ◽  
Inhaled Particles ◽  
Cytochemical Analysis ◽  
Pulmonary Macrophages ◽  
Integrated Screening

There is a great need for the development of a rapid and reliable bioassay to evaluate the pulmonary toxicity of inhaled particles. A number of methods have been proposed, including lung clearance studies, bronchoalveolar lavage analysis, and in vitro cytotoxicity tests. These methods are often limited in scope inasmuch as they measure only one dimension of the pulmonary response to inhaled, instilled or incubated dusts. Accordingly, a comprehensive approach to lung toxicity studies has been developed.To validate the method, rats were exposed for 6 hours or 3 days to various concentrations of either aerosolized alpha quartz silica (Si) or carbonyl iron (CI) particles. Cells and fluids from groups of sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, LDH and protein values were measured in BAL fluids at several time points postexposure. Cells were counted and evaluated for viability, as well as differential and cytochemical analysis. In addition, pulmonary macrophages (PM) were cultured and studied for morphology, chemotaxis, and phagocytosis by scanning electron microscopy.

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