Etest Cannot Be Recommended for In Vitro Susceptibility Testing of Mucorales

Abstract

e Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales species in vitro, while the activities of posaconazole were more restricted. Mucormycoses are rapidly progressing and frequently lethal infections caused by fungi associated with the order Mucorales (1). Rhizopus is the most commonly found genus, followed by the genera Lichtheimia and Mucor (1). Antifungal treatment options consist of lipid formulations of amphotericin B (AMB) as the first-line therapy (2) and posaconazole (PSC) as salvage therapy (3, 4). The rising number of breakthrough mucormycoses (5-7) has stimulated interest in antifungal susceptibility testing (AST) of Mucorales. Phylogenetic studies found that Mucorales embrace a very heterogeneous group of fungi, with their evolutionary distances mirrored in various levels of intrinsic antifungal susceptibilities (8, 9). The recommended international standard methods of the CLSI (Clinical and Laboratory Standards Institute) (10), and EUCAST (European Committee for Antimicrobial Susceptibility Testing) (11) are based on broth microdilution assays. However, ready-to-use commercial tests, such as the Etest, are popular for routine application, as they are simpler to perform and more time efficient (12). To use and exchange data generated with the Etest in routine laboratories, an evaluation of this commercial test compared to a gold standard method is needed.The aims of the present study were 2-fold: to determine amphotericin B and posaconazole susceptibility patterns for the most frequent agents of mucormycoses and to compare the results obtained with Etest and EUCAST.The strain set comprised 131 Mucorales; the clinical strains were collected between 2008 and 2014 in routine laboratory testing at the University Hospital of Innsbruck (see Table S2 in the supplemental material), and an additional 38 reference strains were gained from the Fungal Biodiversity Centre (CBS, Utrecht, The Netherlands) (see Table S3). The strains were cultured on supplementary minimal medium (SUP) agar (13) at 37°C (except that Mucor spp. were grown at 30°C) for 3 to 5 days and preidentified to genus level by micromorphological (Olympus CX21 microscope; Olympus, USA) and macromorphological (Axioplan microscope; Zeiss, Germany) characteristics (6, 14). Species were determined by internal transcribed spacer (ITS) direct sequencing (15). Mycelium was harvested for genomic DNA extraction as previously described (16). ITS sequences were identified with BLAST comparative nucleic acid sequence analysis of sequences in the NCBI database (www.ncbi.nlm.nih.gov/BLAST).In vitro antifungal susceptibilit...

Update from the Laboratory

“…117,118 In addition, a recent study reported poor overall agreement between Etest and EUCAST MICs for amphotericin B and posaconazole (75.1%) when used to measure activity against members of the order Mucorales, the causative agents of mucormycosis, and recommended that the Etest assay not be used against these fungi. 119 The quality and reliability of Etest MIC results may also be influenced by operator technique in applying the strips as well as the trailing affect that may be observed with the azoles against some yeast species. Similar to what has been observed with the YeastOne colorimetric assay, 1 study has also reported a high incidence of C glabrata and C krusei isolates with intermediate caspofungin MICs with this assay.…”

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

“…The Mucorales strain sets used to evaluate the performance of the rnl marker given in Tables S1 and S2 were characterized in a previous study [18]. Fungal stocks stored in 0.9% sodium chloride at −20 • C served as inoculum sources.…”

“…Identification (ID) at the genus level was performed by visualization of microscopic (Olympus CX21 microscope; Olympus, USA) and macroscopic (Axioplan microscope; Zeiss, Germany) characteristics. Species ID was obtained by direct sequencing of the internal transcribed spacer (ITS) [20] with slightly modified primers [18]. Genomic DNA was extracted [21] and ITS sequences were identified using the pairwise sequence alignment tool of the central bureau schimmelcultures (CBS) Knaw database (http://www.cbs.knaw.nl/Collections).…”

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

“…other members of the Mucorales, which could be due to the ignorance regarding the two varieties in the past (Caramalho et al, 2015).…”

“…The choice of antifungal agents used differs widely between various mucoralean fungi at species or even variety level; e.g. amphotericin B shows different activities against Rhizopus microsporus and Rhizopus arrhizus, thus confirming different susceptibility patterns between species or even varieties of mucoralean fungi (Chowdhary et al, 2014;Caramalho et al, 2015).…”