Azole resistance screening in
Aspergillus fumigatus sensu stricto
using the azole-containing agar method (EUCAST E.Def 10.2): conidial suspension filtration and inoculum adjustment before inoculum preparation may not be needed
Authors: Serrano-Lobo Julia , Reigadas Elena , Muñoz Patricia , Escribano Pilar , Guinea Jesús ,   on behalf of the ASPEIN Study Group, Romera Waldo Sánchez-Yebra , Sánchez-Gómez Juan Antonio , Lozano Inmaculada Guerrero , Tejero-García Rocío , Martínez Fernando Cobo , Viñuela Laura , de Luna Francisco Franco-Álvarez , Oya Ana Lara , Moreno-Torres Isabel Casanovas , Ruiz María Pilar Bermúdez , Barrenechea Ana Isabel Suárez , Méndez Carmen Castro , de Pipaón Maite Ruiz Pérez , Condado Héctor Condado , Sánchez Rocío Cebollada , Gómez Concepción López , Romero Emilio David Valverde , Pérez Henar Villar , Temprano Marta Arias , Martínez Alba Guiu , Peláez Teresa , Castello-Abietar Cristian , Giner Carmen Collado , Cruixent Eva Alcoceba , Escobar Carla Iglesias , Lafuente Ana Patricia Martínez , de Torrella Ana Gual , López-Soria Leyre , Jiménez Olga Bello , Barañano Miren Josebe Unzaga , Ramos Iris Sharon Pérez , Albizu Miren Nekane Azkue , Fernández-Torres Marina , Luengo Raúl Gilarranz , Martínez Isabel de Miguel , Martínez Ángeles Sampere , Gutiérrez Silvia Rosa Campos , Mesones María Pía Roiz , Serra Julia Lozano , Serrano Cristina Colmenarejo , Casero Quilez Malo , Zurita María Elena Rodríguez , Barrena Rosa Jiménez , Esgueva Marta Fernández , Santos Oscar Fraile , Sánchez Beatriz Pascual , Echeverría Cristina Labayru , De Freitas Lisbeth Gonçalves , Gay Laura Milian , Fernández Raquel Calleja , Fonseca Alicia Barreales , Puertas Almudena Silvia Tinajas , López-Ramos Irene , Bravo Moisés García , Bellido Juan Luis Muñoz , Andrés Noelia Arenal , Yánez Cristina Veintimilla , Real Susana Hernando , Pérez M. Rosario Ibáñez , Aparicio Federico Miguel Becerra , López Jesús Martínez , Soria Pilar , Mansilla Carmen Aldea , Vidal Belén Lorenzo , Velasco Irene Merino , Gil Mercedes Martín , Gómez María Antonia Miguel , Mestanza Cristina López , Cornejo Jaime Martín , Calle María Luz Asensio , Torres Adrián Antuori , Burgos Eva Cuchí , Sánchez-Reus Ferran , López-Querol Cristina , Para Jaume Juanola , Reverté Francesc Marco , Espasa Mateu , Ardite Josefina Ayats , Olmos Roberto , Ramos David Sanchez , Gómez María Teresa Martín , Palau Montserrat Colomer , Sánchez Iván Prats , Bajador Isabel Pujol , Pernas Antonio Casabella , Bertomeu Frederic Francesc Gómez , Granell Sergio Pardo , Plana Ester Picó , Torres María Dolores Guerrero , Moreno María Mar Olga Pérez , Rodríguez Eva del Corral , Olivares Miguel Fajardo , Pacheco Carmen Pazos , Rodríguez-Mayo María Dolores , Lage Susana Méndez , Domínguez Javier Alba , Fernández Yolanda Vila , Maseda y Susana Orol , Lama Ana Cid , Rey Ana María Rodríguez , González María de los Ángeles Pallarés , Carballo-Fernández Raquel , Pena Rocío Trastoy , Martínez-Lamas Lucía , Blanco Luis Miguel Soria , Pérez-Ayala Ana , de la Pedrosa Elia Gómez García , Sánchez-Romero Isabel , Fernández Sarela García-Masedo , Guinea Jesus , Escribano Pilar , Núñez Ana Gómez , Leiva José González , Palomares Laura Pérez , Liaudansdanskyte Asta Kastanauskaite , Rodríguez Natalia Díaz , Pérez Sarah Montero , Serrano-Lobo Julia , Reigadas Elena , de Paredes Patricia Muñoz García , Melero Inmaculada Quiles , Clemente Paloma García , Durán-Valle María Teresa , Gadea Ignacio , García Marta Martín , Zurita Nelly , Gutiérrez Ainhoa , Cuétara María Soledad , Amador Paloma Merino , de la Torre Irene Díaz , Fernández-Alonso Mirian , Montoro Irene Arco , García-Figueres Eugenio , Hellín Victoria Sánchez , Ros María Isabel Gascón , Borja Salvador Raga , Martín Amparo Valentín , Gaitán Alba Ruiz , Rodríguez Javier Colomina , Olea Beatriz , Clari María de los Ángeles 
Published: May 2024

Azole resistance screening in
Aspergillus fumigatus sensu stricto
can be routinely carried out by using azole-containing agar plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculum preparation are time-consuming. We evaluated whether skipping the filtration and inoculum adjustment steps negatively influenced the performance of the E.Def 10.2 procedure.
A. fumigatus sensu stricto
isolates (
n
= 98), previously classified as azole susceptible or azole resistant (E.Def 9.4 method), were studied. Azole-resistant isolates had either the wild-type
cyp51A
gene sequence (
n
= 1) or the following
cyp51A
gene substitutions: TR
34
-L98H (
n
= 41), G54R (
n
= 5), TR
46
-Y121F-T289A (
n
= 1), or G448S (
n
= 1). In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions obtained by adding distilled water (Tween 20 0.1%) were either filtered and the inocula adjusted to 0.5 McFarland or left unfiltered and unadjusted. Agreements between the agar screening methods using inocula prepared by each procedure were high for itraconazole (99%), voriconazole (100%), and posaconazole (94.9%). Sensitivity and specificity (considering the susceptibility category as per the microdilution E.Def 9.4 method as the gold standard) of E.Def 10.2 were 100% to rule in or rule out resistance when unfiltered and unadjusted suspensions were used; the resistance phenotype of isolates harboring the TR
34
-L98H, G54R, or TR
46
-Y121F-T289A substitutions was correctly detected. Unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in
A. fumigatus sensu stricto
.

IMPORTANCE

Azole resistance screening in
Aspergillus fumigatus sensu stricto
can be routinely carried out by using azole-containing plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculation of plates are time-consuming. We, here, showed that unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in
A. fumigatus sensu stricto
.