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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01083-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 9

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.

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Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

Medical Mycology ◽

10.1093/mmy/myz032 ◽

2019 ◽

Vol 58 (1) ◽

pp. 54-60 ◽

Cited By ~ 1

Author(s):

Beatriz Bustamante ◽

Luis Ricardo Illescas ◽

Andrés Posadas ◽

Pablo E Campos

Keyword(s):

Aspergillus Fumigatus ◽

Latin American ◽

Pcr Amplification ◽

Sensu Stricto ◽

Clinical Isolates ◽

Azole Resistance ◽

Molecular Testing ◽

Naive Patient ◽

In Vitro Susceptibility

Abstract Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5 μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01250-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 19

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.

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Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa316 ◽

2020 ◽

Vol 75 (11) ◽

pp. 3181-3188

Author(s):

Maria Siopi ◽

Olga Rivero-Menendez ◽

Georgios Gkotsis ◽

Anthi Panara ◽

Nikolaos S Thomaidis ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Sensu Stricto ◽

Resistant Isolate ◽

Azole Resistance ◽

Medical Settings ◽

Nationwide Surveillance ◽

The Mean ◽

The One ◽

Organically Grown ◽

Of Greece

Abstract Background Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Methods Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. Results No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06–1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil’s load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). Conclusions This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

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Is Azole Resistance in Aspergillus fumigatus a Problem in Spain?

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02487-12 ◽

2013 ◽

Vol 57 (6) ◽

pp. 2815-2820 ◽

Cited By ~ 58

Author(s):

Pilar Escribano ◽

Teresa Peláez ◽

Patricia Muñoz ◽

Emilio Bouza ◽

Jesús Guinea

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Cryptic Species ◽

Antifungal Susceptibility ◽

Single Species ◽

Azole Resistance ◽

Tubulin Gene ◽

Complex Species ◽

Content Type ◽

Β Tubulin

ABSTRACTAspergillus fumigatuscomplex comprisesA. fumigatusand other morphologically indistinguishable cryptic species. We retrospectively studied 362A. fumigatuscomplex isolates (353 samples) from 150 patients with proven or probable invasive aspergillosis or aspergilloma (2, 121, and 6 samples, respectively) admitted to the hospital from 1999 to 2011. Isolates were identified using the β-tubulin gene, and only 1 isolate per species found in each sample was selected. Antifungal susceptibility to azoles was determined using the CLSI M38-A2 procedure. Isolates were considered resistant if they showed an MIC above the breakpoints for itraconazole, voriconazole, or posaconazole (>2, >2, or >0.5 μg/ml). Most of the samples yielded only 1 species (A. fumigatus[n= 335],A. novofumigatus[n= 4],A. lentulus[n= 3],A. viridinutans[n= 1], andNeosartorya udagawae[n= 1]). The remaining samples yielded a combination of 2 species. Most of the patients were infected by a single species (A. fumigatus[n= 143] orA. lentulus[n= 2]). The remaining 5 patients were coinfected with multipleA. fumigatuscomplex species, althoughA. fumigatuswas always involved; 4 of the 5 patients were diagnosed in 2009 or later. Cryptic species were less susceptible thanA. fumigatus. The frequency of resistance amongA. fumigatuscomplex andA. fumigatusto itraconazole, voriconazole, and posaconazole was 2.5 and 0.3%, 3.1 and 0.3%, and 4.2 and 1.8%, respectively, in the per-isolate analysis and 1.3 and 0.7%, 2.6 and 0.7%, and 6 and 4% in the per-patient analysis. Only 1 of the 6A. fumigatusisolates in which thecyp51Agene was sequenced had a mutation at position G448. The proportion of patients infected by azole-resistantA. fumigatusisolates was low.

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Growth and gliotoxin production by feed-borne Aspergillus fumigatus sensu stricto strains under different interacting environmental conditions

World Mycotoxin Journal ◽

10.3920/wmj2013.1629 ◽

2015 ◽

Vol 8 (1) ◽

pp. 75-85 ◽

Cited By ~ 3

Author(s):

G.A. Pena ◽

M.P. Monge ◽

M.F. Landa ◽

A.M. Dalcero ◽

C.A.R. Rosa ◽

...

Keyword(s):

Growth Rate ◽

Aspergillus Fumigatus ◽

Oxygen Tension ◽

Environmental Conditions ◽

Fungal Growth ◽

Opportunistic Pathogen ◽

Sensu Stricto ◽

Lag Phase ◽

Animal Feeds ◽

Ph Level

In this study the effects of temperature, oxygen tension, water activity (aW), pH, incubation time and their interactions on (1) the lag phase prior to growth, (2) growth rate and (3) gliotoxin production of two feed-borne Aspergillus fumigatus sensu stricto strains, isolated from fermented maize silage and brewer's grains, were evaluated on an agar medium based on these substrates. Regardless of oxygen tension, the growth rate of the two strains decreased significantly as temperature and aW decreased (P<0.05). The optimum conditions for A. fumigatus growth were 37 °C, 0.98 aW for both strains at reduced oxygen tension, regardless of pH level (P<0.05). The studied A. fumigatus strains were able to grow under several incubation conditions, some of them prevalent in stored animal feeds. Some specific interactions that allowed accumulation of gliotoxin at high levels were found. This study showed that gliotoxin production occurred at more restricted conditions than fungal growth. This fact is important, as by maintaining the appropriate conditions in animal feeds, A. fumigatus growth and gliotoxin production can be prevented. In this study, growth rates, lag phases prior to growth and gliotoxin production over a range of environmental conditions provide useful information that can help in predicting the possible fungal contamination of fermented animal feeds. Furthermore, the information is relevant since A. fumigatus is an opportunistic pathogen found in cereals and fermented animal feeds and represents a high risk of contamination to animals and farm workers who handle them improperly.

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Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

Journal of Fungi ◽

10.3390/jof6010011 ◽

2020 ◽

Vol 6 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

Raquel Sabino ◽

Helena Simões ◽

Cristina Veríssimo

Keyword(s):

Aspergillus Fumigatus ◽

Multiplex Pcr ◽

Real Time ◽

Fungal Infections ◽

Sensu Stricto ◽

Azole Resistance ◽

Tissue Samples ◽

Pcr Multiplex ◽

Resistance Patterns ◽

Panfungal Pcr

Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04469-14 ◽

2015 ◽

Vol 59 (3) ◽

pp. 1738-1744 ◽

Cited By ~ 15

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.

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Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program

Journal of Fungi ◽

10.3390/jof7010028 ◽

2021 ◽

Vol 7 (1) ◽

pp. 28

Author(s):

Raquel Sabino ◽

Paulo Gonçalves ◽

Aryse Martins Melo ◽

Daniela Simões ◽

Mariana Oliveira ◽

...

Keyword(s):

Cryptic Species ◽

Environmental Samples ◽

Reference Method ◽

Sensu Stricto ◽

Antifungal Drugs ◽

Surveillance Program ◽

Azole Resistance ◽

Correct Identification ◽

Reference Laboratory ◽

Minimal Inhibitory Concentrations

Identification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017–2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012–2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.

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Azole and Amphotericin B MIC Values against Aspergillus fumigatus: High Agreement between Spectrophotometric and Visual Readings Using the EUCAST EDef 9.3.2 Procedure

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01693-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01693-20 ◽

Cited By ~ 1

Author(s):

Julia Serrano-Lobo ◽

Ana Gómez ◽

Waldo Sánchez-Yebra ◽

Miguel Fajardo ◽

Belén Lorenzo ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Amphotericin B ◽

Fungal Growth ◽

Sensu Stricto ◽

Resistance Rate ◽

Wild Type ◽

Growth Reduction ◽

High Agreement ◽

Content Type

ABSTRACTThe EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MIC readings of azoles and amphotericin B against Aspergillus fumigatussensu lato isolates. A total of 847 A. fumigatussensu lato isolates (A. fumigatus sensu stricto [n = 828] and cryptic species [n = 19]) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatussensu lato isolates may be a convenient alternative to visual endpoint readings.

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